Assay and related methods, kits and devices

Abstract

The present disclosure is related to correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a graft function deterioration in the recipient.

Description

Attorney Docket No.132417-0002WO01 ASSAY AND RELATED METHODS, KITS AND DEVICES CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. provisional patent application App. No. 63 / 441,751, filed on January 27, 2023, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," and U.S. provisional patent application App. No.63 / 532,655, filed on August 14, 2023, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," each of which is incorporated herein by this reference in its entirety. BACKGROUND

[0002] End-stage kidney disease (ESKD) is a leading cause of morbidity and mortality and a major global health burden. To date, kidney transplantation remains to be the most preferred treatment for ESKD, as it provides better quality of life and is cost-effective compared to other kidney replacement therapy modalities. SUMMARY

[0003] This Summary is provided to introduce a selection of concepts in simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key aspects or essential aspects of the claimed subject matter.

[0004] All features of exemplary embodiments which are described in this disclosure and are not mutually exclusive can be combined with one another. Elements of one embodiment can be utilized in the other embodiments without further mention. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with any accompanying Figures.

[0005] The present disclosure may be related to a method of preparing an assay, which may comprise providing a first solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine fragment, to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a graft function deterioration in the recipient, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg, indicates a higher likelihood of the graft function deterioration over a period of aboutAttorney Docket No.132417-0002WO01 ten years or less, and wherein the first solution and the second solution may be the same solution or different solution from each other.

[0006] In some embodiments, the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration.

[0007] In some embodiments, the higher likelihood may be higher than a lower likelihood of the graft function deterioration over the period of about ten years or less in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0008] In some embodiments, the graft function deterioration may be indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

[0009] In some embodiments, the graft function deterioration may be indicated by a decline in estimated Glomerular Filtration Rate (eGFR).

[0010] In some embodiments, the interacting with the Fetuin A fragment may include binding to the Fetuin A fragment.

[0011] In some embodiments, the interacting with the urinary creatinine may include binding to the urinary creatinine.

[0012] In some embodiments, at least one reagent among the first reagent and the second reagent may include an antibody.

[0013] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.3 ng / mg.Attorney Docket No.132417-0002WO01

[0014] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 75 ng / mg.

[0015] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 95 ng / mg.

[0016] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 13 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 12 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 11 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 10 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.2 ng / mg.Attorney Docket No.132417-0002WO01

[0017] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.2 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.1 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 6 ng / mg.

[0018] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.5 ng / mg.

[0019] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 8 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 16 % or higher.

[0020] In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 22 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 32 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 36 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 45 % or higher.Attorney Docket No.132417-0002WO01 In some embodiments, the higher likelihood of the graft function deterioration may be about 50 % or higher.

[0021] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % to about 50%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 35%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 20%. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % to about 25%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % to about 50%.

[0022] In some embodiments, the graft function deterioration occurs after 2 years. In some embodiments, the graft function deterioration occurs after 3 years. In some embodiments, the graft function deterioration occurs after 4 years. In some embodiments, the graft function deterioration occurs after 5 years. In some embodiments, the graft function deterioration occurs after 6 years. In some embodiments, the graft function deterioration occurs after 7 years. In some embodiments, the graft function deterioration occurs after 8 years. In some embodiments, the graft function deterioration occurs after 9 years. In some embodiments, the graft function deterioration occurs within 7 years. In some embodiments, the graft function deterioration occurs within 8 years. In some embodiments, the graft function deterioration occurs within 9 years.

[0023] In some embodiments, the method may further comprise assessing the probability of the graft function deterioration, based on the ratio and at least one other marker.

[0024] In some embodiments, the at least one other marker may comprise age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof.

[0025] In some embodiments, the assessed probability may exhibit at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may exhibit at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 95 percent (%) of the AUC.Attorney Docket No.132417-0002WO01

[0026] The present disclosure may be related to an assay kit to determine a likelihood of a progressive renal decline, in which the assay kit may comprise a first solution including a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine fragment to indicate the level of the Fetuin A fragment in a urine sample from a recipient of a kidney transplantation after the kidney transplantation; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg20 ng / mg indicates a higher likelihood of the graft function deterioration over a period of about ten years or less, and wherein the first solution and the second solution may be the same solution or different solution from each other.

[0027] In some embodiments, the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration.

[0028] In some embodiments, the higher likelihood may be higher than a lower likelihood of the graft function deterioration over the period of about ten years or less in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0029] In some embodiments, the graft function deterioration may be indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

[0030] In some embodiments, the graft function deterioration may be indicated by a decline in estimated Glomerular Filtration Rate (eGFR).

[0031] In some embodiments, the interacting with the Fetuin A fragment may include binding to the Fetuin A fragment.

[0032] In some embodiments, the interacting with the urinary creatinine may include binding to the urinary creatinine.

[0033] In some embodiments, at least one reagent among the first reagent and the second reagent may include an antibody.

[0034] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27Attorney Docket No.132417-0002WO01 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.3 ng / mg.

[0035] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 75 ng / mg.

[0036] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 95 ng / mg.Attorney Docket No.132417-0002WO01

[0037] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 13 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 12 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 11 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 10 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.2 ng / mg.

[0038] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.2 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.1 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.5 ng / mg.

[0039] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 8 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 16 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 22 % or higher. In some embodiments, theAttorney Docket No.132417-0002WO01 higher likelihood of the graft function deterioration may be about 25 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 32 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 36 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 45 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 50 % or higher.

[0040] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % to about 50%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 35%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 20%. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % to about 25%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % to about 50%.

[0041] In some embodiments, the graft function deterioration occurs after 2 years. In some embodiments, the graft function deterioration occurs after 3 years. In some embodiments, the graft function deterioration occurs after 4 years. In some embodiments, the graft function deterioration occurs after 5 years. In some embodiments, the graft function deterioration occurs after 6 years. In some embodiments, the graft function deterioration occurs after 7 years. In some embodiments, the graft function deterioration occurs after 8 years. In some embodiments, the graft function deterioration occurs after 9 years. In some embodiments, the graft function deterioration occurs within 7 years. In some embodiments, the graft function deterioration occurs within 8 years. In some embodiments, the graft function deterioration occurs within 9 years.

[0042] In some embodiments, the device may be to determine the level of the Fetuin A fragment and the level of the urinary creatinine, to assess the probability of the graft function deterioration, based on the ratio and at least one other marker.

[0043] In some embodiments, the at least one other marker may comprise age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtrationAttorney Docket No.132417-0002WO01 rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof.

[0044] In some embodiments, the assessed probability may exhibit at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may exhibit at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 95 percent (%) of the AUC.

[0045] The present disclosure may be related to a method of correlating a likelihood of a graft function deterioration, in which the method may comprise determining the level of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplantation after the kidney transplantation, wherein the first biomarker may be a fragment of a post-translationally modified Fetuin-A fragments in urine fragment, wherein the second biomarker may be a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a likelihood of a graft function deterioration in the recipient over a period of about ten years, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg indicates a higher likelihood of the graft function deterioration over the period of about ten years or less.

[0046] In some embodiments, the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration.

[0047] In some embodiments, the higher likelihood may be higher than a lower likelihood of the graft function deterioration over the period of about ten years or less in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0048] In some embodiments, the graft function deterioration may be indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

[0049] In some embodiments, the graft function deterioration may be indicated by a decline in estimated Glomerular Filtration Rate (eGFR).

[0050] In some embodiments, the interacting with the Fetuin A fragment may include binding to the Fetuin A fragment.

[0051] In some embodiments, the interacting with the urinary creatinine may include binding to the urinary creatinine.Attorney Docket No.132417-0002WO01

[0052] In some embodiments, at least one reagent among the first reagent and the second reagent may include an antibody.

[0053] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.3 ng / mg.

[0054] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 75 ng / mg.

[0055] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 91Attorney Docket No.132417-0002WO01 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 95 ng / mg.

[0056] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 13 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 12 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 11 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 10 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.2 ng / mg.

[0057] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.2 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.1 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.5 ng / mg.

[0058] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may beAttorney Docket No.132417-0002WO01 about 8 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 16 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 22 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 32 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 36 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 45 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 50 % or higher.

[0059] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % to about 50%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 35%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 20%. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % to about 25%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % to about 50%.

[0060] In some embodiments, the graft function deterioration occurs after 2 years. In some embodiments, the graft function deterioration occurs after 3 years. In some embodiments, the graft function deterioration occurs after 4 years. In some embodiments, the graft function deterioration occurs after 5 years. In some embodiments, the graft function deterioration occurs after 6 years. In some embodiments, the graft function deterioration occurs after 7 years. In some embodiments, the graft function deterioration occurs after 8 years. In some embodiments, the graft function deterioration occurs after 9 years. In some embodiments, the graft function deterioration occurs within 7 years. InAttorney Docket No.132417-0002WO01 some embodiments, the graft function deterioration occurs within 8 years. In some embodiments, the graft function deterioration occurs within 9 years.

[0061] In some embodiments, the method may further comprise assessing the probability of the graft function deterioration, based on the ratio and at least one other marker.

[0062] In some embodiments, the at least one other marker may comprise age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof.

[0063] In some embodiments, the assessed probability may exhibit at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may exhibit at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 95 percent (%) of the AUC. BRIEF DESCRIPTION OF THE FIGURES

[0064] Figure 1 illustrates a Kaplan-Meier Survival Curve for kidney transplant recipients (KTRs) with death-censored graft failure of Primary Set according to a non-limiting embodiment.

[0065] Figure 2A illustrates a Kaplan-Meier curve for KTRs in the Primary Set with (a) non- Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0066] Figure 2B illustrates a Kaplan-Meier curve for KTRs in the Primary Set with (b) Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0067] Figure 3 illustrates a Kaplan-Meier Survival Curve for KTRs with death-censored graft failure of Validation Set, according to a non-limiting embodiment.

[0068] Figure 4A illustrates a Kaplan-Meier curve for subjects in the Validation Set with (a) non- Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0069] Figure 4B illustrates a Kaplan-Meier curve for subjects in the Validation Set with (b) Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0070] Figure 5 illustrates a Kaplan-Meier Survival Curve for 635 KTRs with death-censored graft failure, according to a non-limiting embodiment.

[0071] Figure 6A illustrates a Kaplan-Meier curve for KTRs in the 24h Set with (a) non-Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0072] Figure 6B illustrates a Kaplan-Meier curve for KTRs in the 24h Set with (b) Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment.

[0073] Figure 7 illustrates a Receiver-operating characteristic (ROC) curve regarding uPTM- FetA / uCr and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.Attorney Docket No.132417-0002WO01

[0074] Figure 8 illustrates a Receiver-operating characteristic (ROC) curve regarding a combination of uPTM-FetA / uCr, age, sex, time, and UACR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0075] Figure 9 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time, UACR, and eGFR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0076] Figure 10 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time, UACR, eGFR, and the presence of human leucocyte antigen class II antibodies (HLA II) and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0077] Figure 11 illustrates a Receiver-operating characteristic (ROC) curve regarding uPTM- FetA / uCr and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0078] Figure 12 illustrates a Receiver-operating characteristic (ROC) curve regarding a combination of uPTM-FetA / uCr, age, sex, time, and UACR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0079] Figure 13 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time, UACR, and eGFR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0080] Figure 14 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time, UACR, eGFR, and the presence of human leucocyte antigen class II antibodies (HLA II) and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0081] Figure 15 illustrates a flowchart of the study population selection, according to a non-limiting embodiment.

[0082] Figure 16 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the serum creatine level, according to a non-limiting embodiment.

[0083] Figure 17 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the eGFR, according to a non-limiting embodiment.

[0084] Figure 18 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the 24 hour albumin excretion, according to a non-limiting embodiment.

[0085] Figure 19 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the 24 hours urinary protein excretion, according to a non-limiting embodiment.Attorney Docket No.132417-0002WO01

[0086] Figure 20 illustrates Kaplan-Meier curve for death-censored graft failure below and above the median of 24 h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment.

[0087] Figure 21 illustrates Kaplan-Meier curve for death-censored graft function deterioration below and above the median of 24h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment.

[0088] Figure 22 illustrates a Kaplan-Meier curve for all-cause mortality below and above the median of 24h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment.

[0089] Figure 23 illustrates a flowchart of the study participants selection according to a non-limiting embodiment;

[0090] Figure 24 illustrates Kaplan-Meier analysis for death-censored graft function deterioration survival per tertile of 24h uC-FetA excretion according to a non-limiting embodiment; and

[0091] Figure 25 illustrates a Forest plot for the association of uC-FetA with death-censored graftfunction deterioration insubgroupsaccording to a non-limiting embodiment.

[0092] Figure 26 illustrates Kaplan-Meier analysis for patient survival per tertile of 24h uC-FetA excretion according to a non-limiting embodiment.

[0093] Figure 27 illustrates a calibration curve of the Human uPTM3-DKD ELISA (calibration range is 7.813 - 500 ng / mL) according to a non-limiting embodiment. DETAILED DESCRIPTION OF THE DISCLOSURE

[0094] The present disclosure is based on unexpected discoveries that a urine protein and its fragment, post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), is differentially presented in a kidney transplant recipient (KTR) with a higher likelihood of a graft function deterioration. These protein molecules are therefore useful markers for diagnosing a graft function deterioration at different stage including an early stage. Therefore, uPTM-FetA has been identified as a biomarker for acute kidney injury and in extension thereof, is proposed as a biomarker for early detection of kidney function decline.

[0095] To date, kidney transplantation remains to be the most preferred treatment for ESKD, as it provides better quality of life and is most cost-effective compared to other kidney replacement therapy modalities. However, despite this success, a kidney transplant recipient (KTR) can remain at risk of graft functional loss and at risk of morbidity and premature mortality, which is largely attributable to deterioration of kidney graft function. Looking at the patient's perspective, survival of the kidney graft is acknowledged to be more important than life itself, and the patients would rather die than return to dialysis. Thus, more efforts should be made to detect graft functional loss earlier so that appropriate treatment can be given and graft failure can be prevented. In the current clinical setting, routine evaluation of graft function is based on serum creatinine and proteinuria. In the casesAttorney Docket No.132417-0002WO01 with marked serum creatinine elevation or the presence of proteinuria, a kidney biopsy is considered. Unfortunately, these evaluations are imperfect, especially regarding the prognosis prediction of the graft. Therefore, there is a need for biomarkers that can be used to identify KTR at risk of graft function deterioration and subsequent graft failure such as alternative non-invasive markers that are able to independently identify patients at risk of graft function deterioration and subsequent graft failure.

[0096] As used herein, the term "medical intervention" for the deterioration of kidney graft function in a KTR means a treatment the recipient in attempt to stabilize, slow down, delay, or suppress the deterioration, which may result in a decrease in the rate of the deterioration in the recipient. Various types of medical interventions can be implemented. Examples of medical intervention include guided diets, restrictive diets, limiting sodium and other ion intake, reducing or avoiding alcohol consumption, reducing or avoiding smoking cigarette or consuming related products, intaking of various medications, managing weight, kidney transplant or retransplant, dialysis, other medically recognizable treatments, and any combination thereof.

[0097] As used herein, the term "average Fetuin A fragment excretion" means the amount of the excretion of Fetuin A fragment, such as Urinary Connecting peptide containing Fetuin-A, to a urine averaged over a predetermined period of time. For example, the average Fetuin A fragment excretion can be averaged over 2 to 48 hours or longer, or shorter.

[0098] Accordingly, the present disclosure is related to a diagnostic method using a biomarker, which can be a protein or a protein that can be considered as a fragment of the protein. A urine sample, a serum sample, or both the urine and serum samples, can be collected from a subject and the urine level, serum level or both levels of the biomarker can be determined via various methods, e.g., mass spectrometry and immune analysis.

[0099] When a biomarker contains a single protein molecule, its level in a subject can be compared with a reference point to determine a corresponding indication or likelihood of a target condition. The reference point, representing the level of the same biomarker such as a level of a biomarker in a normal subject without a substantial deterioration of kidney graft function in a KTR, can be determined based on the representative levels of the biomarker in groups of KTRs. For example, it can be the middle point between the mean levels of these two groups. A biomarker level higher than the reference point is indicative of the target condition.

[0100] There is a significant clinical need for novel markers to predict a kidney function decline in patients with ESKD or KTRs. A post-translationally modified Fetuin-A fragments in urine fragments (uPTM-FetA) can be a biomarker to indicate indicates a likelihood of the renal decline to the kidney endpoint and can predict a kidney function decline in patients with ESKD or KTRs.

[0101] Fetuin-A (also known as alpha-2-Heremans-Schmid glycoprotein) is a heterodimeric glycoprotein with 367 amino acid sequences containing 18 amino acids of a signal peptide, 282 aminoAttorney Docket No.132417-0002WO01 acids of A-chain, 40 amino acid chains of connecting peptide, and 27 amino acids of B-chain that undergo various post-translational modifications before being secreted outside of the producing cells. Fetuin-A such as a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), which can include a post-translationally modified Fetuin-A fragments in urine fragments (uPTM-FetA) (also known as alpha-2- Heremans–Schmid glycoprotein), has characteristics that suggest that it could serve as such an alternative, independent non-invasive marker. Fetuin-A is elevated in the urine of patients with acute kidney injury (AKI), and recent evidence suggests that it is also linked to (degree of) interstitial fibrosis / tubular atrophy (IFTA). Incidence of AKI and presence of IFTA after kidney transplantation has been known to be associated with unfavorable long-term graft outcomes. Urinary Fetuin-A is also elevated in patients with chronic kidney diseases such as autosomal dominant polycystic kidney disease and focal segmental glomerular sclerosis. Next to that, urinary Fetuin-A has been shown previously to be associated with kidney function decline in patients with chronic kidney disease including diabetic nephropathy, and this protein has been proposed to be used as a biomarker for early detection in these diseases. However, no study has been done to measure urinary Fetuin-A in KTR population and investigate the plausible association with the graft outcome.

[0102] In some embodiments, post-translationally modified Fetuin-A fragments in urine (uPTM- FetA) can be used as a biomarker for acute kidney injury and in extension thereof, is proposed as a biomarker for early detection of kidney function decline. In some embodiments, uPTM-FetA protein can serve as a marker of graft function deterioration in KTR. For example, the level of uPTM-FetA in stable outpatient KTR can be used to determine a likelihood of a higher likelihood of the graft function deterioration over time, such as about ten years or less. In some embodiments, the primary end-point can be death-censored graft function deterioration i.e., the need of re-transplantation or (re- )initiation of dialysis) and the secondary end-point was all- cause mortality.

[0103] Because of the potential pathophysiological roles of urinary Fetuin-A in the occurrence of kidney function decline, the association of urinary Fetuin-A or its fragment with the graft function deterioration can be used to determine a likelihood of the graft function deterioration over time.

[0104] Accordingly, in some embodiments, a method of preparing an assay, e.g., for correlating a likelihood of a graft function deterioration in a recipient of a kidney transplantation can comprise providing a first solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine fragment, to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a graft function deterioration in the recipient, wherein the ratioAttorney Docket No.132417-0002WO01 being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg, indicates a higher likelihood of the graft function deterioration over a period of about ten years or less, wherein the first solution and the second solution are the same solution or different solution from each other.

[0105] Moreover, In some embodiments, an assay kit to determine a likelihood of a progressive renal decline may comprise a first solution including a first reagent to interact with a post- translationally modified Fetuin-A fragments in urine fragment to indicate the level of the Fetuin A fragment in a urine sample from a recipient of a kidney transplantation after the kidney transplantation; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of the graft function deterioration over a period of about ten years or less, wherein the first solution and the second solution are the same solution or different solution from each other.

[0106] Moreover, in some embodiments, a method of correlating a likelihood of a graft function deterioration may comprise determining the level of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplantation after the kidney transplantation, wherein the first biomarker may be a fragment of a post-translationally modified Fetuin-A fragments in urine fragment, wherein the second biomarker may be a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a likelihood of a graft function deterioration in the recipient over a period of about ten years, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of the graft function deterioration over the period of about ten years or less.

[0107] In some embodiments, the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration.

[0108] In some embodiments, the higher likelihood may be higher than a lower likelihood of the graft function deterioration over the period of about ten years or less in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinineAttorney Docket No.132417-0002WO01 lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0109] In some embodiments, the graft function deterioration may be indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

[0110] In some embodiments, the graft function deterioration may be indicated by a decline in estimated Glomerular Filtration Rate (eGFR).

[0111] In some embodiments, the interacting with the Fetuin A fragment may include binding to the Fetuin A fragment.

[0112] In some embodiments, the interacting with the urinary creatinine may include binding to the urinary creatinine.

[0113] In some embodiments, at least one reagent among the first reagent and the second reagent may include an antibody.

[0114] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 29.3 ng / mg.

[0115] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 65Attorney Docket No.132417-0002WO01 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 75 ng / mg.

[0116] In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg may be higher than about 95 ng / mg.

[0117] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 13 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 12 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 11 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 10 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8.2 ng / mg.

[0118] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.3 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.2 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7.1 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 7 ng / mg. InAttorney Docket No.132417-0002WO01 some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 6 ng / mg.

[0119] In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 5 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 4 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.9 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.8 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.7 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.6 ng / mg. In some embodiments, the second ratio being lower than about 14 ng / mg may be lower than about 3.5 ng / mg.

[0120] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 8 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 16 % or higher.

[0121] In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 22 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 32 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 36 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 45 % or higher. In some embodiments, the higher likelihood of the graft function deterioration may be about 50 % or higher.

[0122] In some embodiments, the higher likelihood of the graft function deterioration may be about 5 % to about 50%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 35%. In some embodiments, the higher likelihood of the graft function deterioration may be about 25 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 10 % to about 20%. In some embodiments, the higher likelihood of the graft function deterioration may be about 15 % to aboutAttorney Docket No.132417-0002WO01 25%. In some embodiments, the higher likelihood of the graft function deterioration may be about 20 % to about 30%. In some embodiments, the higher likelihood of the graft function deterioration may be about 30 % to about 40%. In some embodiments, the higher likelihood of the graft function deterioration may be about 35 % to about 45%. In some embodiments, the higher likelihood of the graft function deterioration may be about 40 % to about 50%.

[0123] In some embodiments, the graft function deterioration occurs after 2 years. In some embodiments, the graft function deterioration occurs after 3 years. In some embodiments, the graft function deterioration occurs after 4 years. In some embodiments, the graft function deterioration occurs after 5 years. In some embodiments, the graft function deterioration occurs after 6 years. In some embodiments, the graft function deterioration occurs after 7 years. In some embodiments, the graft function deterioration occurs after 8 years. In some embodiments, the graft function deterioration occurs after 9 years. In some embodiments, the graft function deterioration occurs within 7 years. In some embodiments, the graft function deterioration occurs within 8 years. In some embodiments, the graft function deterioration occurs within 9 years.

[0124] In some embodiments, the method may further comprise assessing the probability of the graft function deterioration, based on the ratio and at least one other marker.

[0125] In some embodiments, the at least one other marker may comprise age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof.

[0126] In some embodiments, the assessed probability may exhibit at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). In some embodiments, the assessed probability may exhibit at least about 80 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 85 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 90 percent (%) of the AUC. In some embodiments, the assessed probability may exhibit at least about 95 percent (%) of the AUC.

[0127] In some embodiments, the interacting with the Fetuin A fragment includes binding to the Fetuin A. In some embodiments, the reagent includes an antibody.

[0128] uPTM-FetA excretion is associated with kidney function and also with increased markers of proximal tubular injury and 24h urinary protein excretion. Importantly, uPTM-FetA excretion is independently associated with graft function deterioration in stable KTR, particularly in patients with relatively good kidney function.

[0129] In some embodiments, in KTRs, shorter time after transplantation, living donor, older donor age, the use of proliferation inhibitors, and lower AST level can be independently associated with higher uPTM-FetA excretion. In some embodiments, urinary L-FABP excretion, a marker of proximal tubular injury, can also be independently associated with uPTM-FetA excretion. In some embodiments,Attorney Docket No.132417-0002WO01 increased uPTM-FetA excretion can be associated with an increased risk of graft function deterioration even after adjustment with potential confounders. In healthy adults, the kidney does not express Fetuin- A. However, upon injury, proximal tubule epithelial cells (PTEC) is able to express Fetuin-A and release it to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary Fetuin-A can be predominantly present in the urinary exosome fraction instead of from non-exosomal fraction. PTEC can locally produce Fetuin-A under hypoxic conditions after stimulation from hypoxia-inducible transcription factor. It may be that the presence of Fetuin-A in the proximal tubules is aids in protecting the kidney from hypoxia- induced kidney inflammation by preventing the shifting of macrophage to pro-inflammatory macrophage M1, and from hypoxia-induced fibrosis by antagonizing TGF-β signaling.

[0130] The human precursor protein of fetuin A consists of three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a post translational modification, limited proteolysis, after which only the A and B chain form the active fetuin A protein. The monoclonal antibody in the ELISA kit can detect the connecting peptide-containing fetuin A. Therefore, uPTM Fetuin-A detected by this ELISA kit might be the inactive fetuin A without protecting effects of kidney function and elevated uPTM- FetA was associated with risk of eGFR decline in patients. Next to the local production, Fetuin-A in the urine may also be originated from the circulation. Fetuin-A is a negatively charged molecule with a molecular weight of approximately 60 kilodalton, similar to that of albumin. In physiological condition, selective amount of albumin may pass the can pass through the glomerular filtration barrier (GFB) where it will be reabsorbed in the tubular compartment by the proximal tubule epithelial cells (PTEC). As Fetuin-A and albumin have similar properties, equivalent handling by the kidney can be expected. Fetuin-A can be absorbed in the proximal tubule in healthy rat by the megalin-mediated endocytosis, predominantly at the S1 segment and to the lower extent at the S2 segment. In condition where there is an accumulation of injury in the glomerulus, GFB will be disrupted and excessive amounts of different types of plasma proteins will leaked to the tubular compartment. This causes the amount of filtered protein needed to be reabsorbed by the proximal tubules to increase. As the reabsorption capacity of PTEC is not unlimited, protein from the circulation will present in the urine once this mechanism is saturated. Next to that, the filtered proteins may cause injury in the proximal tubules via various mechanisms, i.e. by obstructing tubular lumen with protein casts, by causing energy depletion and lysosomal rupture due to abundant amount of protein overload that needs to be reabsorbed by the PTEC, by triggering PTEC to release pro-inflammatory and pro-fibrotic cytokines, by causing intracellular accumulation of lipidated proteins that will trigger tubular apoptosis, and by activating complement system to cause direct injury to the PTEC. When this occurs, the ability of PTEC to locally produce Fetuin-A and to reabsorb filtered Fetuin-A can be impaired. As uPTM-FetA excretion is significantly associated with an increased risk of graft function deterioration even after adjustment for potentialAttorney Docket No.132417-0002WO01 confounders including urinary protein excretion, the origin of uPTM-FetA can be from the local production by the PTEC instead of from the impaired reabsorption process. In the sensitivity analyses, uPTM-FetA may not be associated with graft function deterioration in patients with eGFR below 30 ml / min / 1.73m2. Fetuin-A from the circulation may also end up in the urine when there is an accumulated injury in the kidney. Thus, uPTM-FetA measured in this subgroup of patients may consists of locally produced Fetuin-A and circulating Fetuin-A. Fetuin-A in the circulation is not associated with kidney function.

[0131] Both acute and chronic kidney injury are tightly associated with the occurrence of hypoxia. Among all structures within the kidney organ, PTEC are the most sensitive and most vulnerable, as these cells are highly active and have high oxygen demand. Because hypoxia plays a major role in the progression of the kidney disease and may present in the relatively early stages of kidney injury even before the structural injury occurred, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion-induced AKI, urinary Fetuin- A excretion increased before the surge of serum creatinine. Moreover, Fetuin-A deposition in the PTEC increased before structural injury in the tubules present morphologically. In type 2 diabetes mellitus patients, urinary Fetuin-A able to display progression of kidney function deterioration earlier than albuminuria. As one of the most important requirements for a biomarker is to reflect an underlying pathophysiology of the disease, in some embodiments, measurement of Fetuin-A in the urine may offer additional benefit as a biomarker over currently used parameters for early detection of graft injury in KTR such as serum creatinine or proteinuria.

[0132] In some embodiments, associating Fetuin-A with at least one other biomarker related to a kidney can be an indicator of the kidney function or its detoriation. For example, associating Fetuin-A with a creatinine can be such an indicator. A kidney function can be judged by the level of filtration that occurs. Filtration is accomplished by the tiny blood vessels in the kidney called "glomeruli". So overall kidney function is called the "glomerular filtration rate" (GFR), measured as the volume of blood filtered per minute (milliters / minute). GFR can vary with other variables, such as age and sex. GFR can be estimated (eGFR) using formulas that take into account your age, sex and a blood test called creatinine. Kidney failure is most often found when the creatinine level is high, indicating that kidney function is reduced. Creatinine is a molecule made by your muscles. A normal kidney will remove creatinine from the blood stream and get rid of it in urine. More creatinine in the blood is a sign that the kidneys aren't cleaning the blood as well as they should. Accordingly, associating Fetuin- A with GFR or creatinine can be a relatively reliable indicator of kidney function deterioration in KTRs.

[0133] ASSAY

[0134] In some embodiments, various methods, reagents, devices, and kits can be used to measure the level of Fetuin-A or a Fetuin-A-based fragment in various mediums, such as in a urine, serum, andAttorney Docket No.132417-0002WO01 other bodily fluids. In some embodiments, the enzyme-linked immunosorbent assay (ELISA) can be used to measure the level of Fetuin-A. For example, the ELISA can be used for quantitative measurement of Fetuin-A with specific post translational modification (PTM) in human urine and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists. For example, calibrators or unknown urine samples are mixed with an antibody having a binding affinity to a part of Fetuin-A, such as anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate pre-bounded with Fetuin-A or its segment, such as unique PTM Fetuin-A. The monoclonal antibody recognizes Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, in some embodiments, signal-generating molecule such as signal-molecule-tagged antibody can be used. In some embodiments, a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3',5,5'- tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.

[0135] In some embodiments, A method of correlating a likelihood of a graft function deterioration in a recipient of a kidney transplantation can comprise obtaining a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0136] In some embodiments, a method of preparing an assay can comprise providing a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0137] In some embodiments, an assay kit to determine a likelihood of a graft function deterioration of a transplanted kidney can comprise a solution including a concentration of a reagent to interact with an amount of a urinary connecting peptide-containing Fetuin A fragment in a urine sample from a recipient of a kidney transplantation. A device can be provided to measure an average Fetuin A fragment excretion to urine from the solution, to correlate the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measuredAttorney Docket No.132417-0002WO01 average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0138] In some embodiments, A method of correlating a likelihood of a graft function deterioration in a recipient of a kidney transplantation can comprise obtaining a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0139] In some embodiments, a method of preparing an assay can comprise providing a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0140] In some embodiments, an assay kit to determine a likelihood of a graft function deterioration of a transplanted kidney can comprise a solution including a concentration of a reagent to interact with an amount of a urinary connecting peptide-containing Fetuin A fragment in a urine sample from a recipient of a kidney transplantation. A device can be provided to measure an average Fetuin A fragment excretion to urine from the solution, to correlate the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0141] the present disclosure is related to a diagnostic method using a biomarker, which can be a protein or a protein that can be considered as a fragment of the protein. A urine sample, a serum sample, or both the urine and serum samples, can be collected from a subject and the urine level, serum level or both levels of the biomarker can be determined via various methods, e.g., mass spectrometry and immune analysis.

[0142] When a biomarker contains a single protein molecule, its level in a subject can be compared with a reference point to determine a corresponding indication or likelihood of a target condition. The reference point, representing the level of the same biomarker such as a level of a biomarker in a normal subject without a substantial deterioration of kidney graft function in a KTR, can be determined based on the representative levels of the biomarker in groups of KTRs. For example, itAttorney Docket No.132417-0002WO01 can be the middle point between the mean levels of these two groups. A biomarker level higher than the reference point is indicative of the target condition.

[0143] Fetuin-A such as Urinary Connecting peptide containing Fetuin-A (uC-FetA) (also known as alpha-2- Heremans–Schmid glycoprotein) has characteristics that suggest that it could serve as such an alternative, independent non-invasive marker. Fetuin-A is elevated in the urine of patients with acute kidney injury (AKI), and recent evidence suggests that it is also linked to (degree of) interstitial fibrosis / tubular atrophy (IFTA). Incidence of AKI and presence of IFTA after kidney transplantation has been known to be associated with unfavorable long-term graft outcomes. Urinary Fetuin-A also elevated in patients with chronic kidney diseases such as autosomal dominant polycystic kidney disease and focal segmental glomerular sclerosis. Next to that, urinary Fetuin-A has been shown previously to be associated with kidney function decline in patients with chronic kidney disease including diabetic nephropathy, and this protein has been proposed to be used as a biomarker for early detection in these diseases. However, no study has been done to measure urinary Fetuin-A in KTR population and investigate the plausible association with the graft outcome.

[0144] In some embodiments, Urinary Connecting peptide containing Fetuin-A (uC-FetA) can be used as a biomarker for acute kidney injury and in extension thereof, is proposed as a biomarker for early detection of kidney function decline. In some embodiments, uC-FetA protein can serve as a marker of graft function deterioration in KTR. For example, the level of uC-FetA in stable outpatient KTR can be used to determine a likelihood of a higher likelihood of the graft function deterioration over time, such as about ten years or less. In some embodiments, the primary end-point can be death- censored graft function deterioration i.e., the need of re-transplantation or (re-)initiation of dialysis) and the secondary end-point was all- cause mortality.

[0145] Because of the potential pathophysiological roles of urinary Fetuin-A in the occurrence of kidney function decline, the association of urinary Fetuin-A or its fragment with the graft function deterioration can be used to determine a likelihood of the graft function deterioration over time.

[0146] Accordingly, in some embodiments, A method of correlating a likelihood of a graft function deterioration in a recipient of a kidney transplantation can comprise obtaining a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0147] In some embodiments, a method of preparing an assay can comprise providing a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantationAttorney Docket No.132417-0002WO01 and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0148] In some embodiments, an assay kit to determine a likelihood of a graft function deterioration of a transplanted kidney can comprise a solution including a concentration of a reagent to interact with an amount of a urinary connecting peptide-containing Fetuin A fragment in a urine sample from a recipient of a kidney transplantation. A device such as an ELISA reader can be provided to measure an average Fetuin A fragment excretion to urine from the solution, to correlate the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less.

[0149] In some embodiments, the excretion being higher than about 0.8 µg / h can trigger a medical intervention to suppress the graft function deterioration.

[0150] In some embodiments, the graft function deterioration can be indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

[0151] In some embodiments, the recipient may have been indicated to have the eGFR of about 25 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher at a time around collecting the urine sample.

[0152] In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 2 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 4 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 6 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 12 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 24 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 36 hours. In some embodiments, the average Fetuin A fragment excretion to the urine can be averaged over no less than about 48 hours.

[0153] In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 1 day after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 2 days after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 1 week after the kidney transplantation. In someAttorney Docket No.132417-0002WO01 embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 2 weeks after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 1 month after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 3 months after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 6 months after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 12 months after the kidney transplantation. In some embodiments, the urine sample can be collected from the recipient of the kidney transplantation at least about 24 months after the kidney transplantation.

[0154] In some embodiments, the e average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about nine years or less. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about seven years or less. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about five years or less. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about three years or less. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about one year or less.

[0155] In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 0.9 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 1 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 2 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 2.1 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 2.2 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 2.3 µg / h. In some embodiments, the average Fetuin A fragment excretion being higher than about 0.8 µg / h can be higher than about 2.5 µg / h.

[0156] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h toAttorney Docket No.132417-0002WO01 about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.5 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.59 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.6 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.7 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.78 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.79 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 1.8 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.1 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.15 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.18 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.2 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.22 or more.

[0157] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be from about 1.5 to about 2.5. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion beingAttorney Docket No.132417-0002WO01 about 0.9 µg / h or lower can be from about 1.6 to about 2.3. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be from about 1.59 to about 2.22.

[0158] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.2 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.26 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.5 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 2.7 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 3 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 3.5 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 3.6 or more.

[0159] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 3.7 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 3.8 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 4 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 4.5 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragmentAttorney Docket No.132417-0002WO01 excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 4.7 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 4.8 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 4.9 or more. In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be about 5 or more.

[0160] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be from about 2 to about 5.

[0161] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be from about 2 to about 4.5.

[0162] In some embodiments, a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower can be from about 2.5 to about 4.5.

[0163] In some embodiments, the interacting with the Fetuin A fragment includes binding to the Fetuin A.

[0164] In some embodiments, the reagent includes an antibody.

[0165] uC-FetA excretion is associated with kidney function and also with increased markers of proximal tubular injury and 24h urinary protein excretion. Importantly, uC-FetA excretion is independently associated with graft function deterioration in stable KTR, particularly in patients with relatively good kidney function.

[0166] In some embodiments, in KTRs, shorter time after transplantation, living donor, older donor age, the use of proliferation inhibitors, and lower AST level can be independently associated with higher 24h uC-FetA excretion. In some embodiments, 24h urinary L-FABP excretion, a marker of proximal tubular injury, can also be independently associated with 24h uC-FetA excretion. In some embodiments, increased uC-FetA excretion can be associated with an increased risk of graft function deterioration even after adjustment with potential confounders. In healthy adults, the kidney does not express Fetuin- A. However, upon injury, proximal tubule epithelial cells (PTEC) is able to express Fetuin-A and release it to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary Fetuin-A can be predominantly present in the urinary exosome fraction instead of from non-exosomal fraction. PTEC can locally produce Fetuin-A under hypoxic conditions after stimulationAttorney Docket No.132417-0002WO01 from hypoxia-inducible transcription factor. It may be that the presence of Fetuin-A in the proximal tubules is aids in protecting the kidney from hypoxia- induced kidney inflammation by preventing the shifting of macrophage to pro-inflammatory macrophage M1, and from hypoxia-induced fibrosis by antagonizing TGF-β signaling.

[0167] The human precursor protein of fetuin A consists of three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a posttranslational modification, limited proteolysis, after which only the A and B chain form the active fetuin A protein. The monoclonal antibody in the ELISA kit can detect the connecting peptide-containing fetuin A. Therefore, uC-FetA detects by this ELISA kit might be the inactive fetuin A without protecting effects of kidney function and elevated uC-FetA was associated with risk of eGFR decline in patients. Next to the local production, Fetuin-A in the urine may also be originated from the circulation. Fetuin-A is a negatively charged molecule with a molecular weight of approximately 60 kilodalton, similar to that of albumin. In physiological condition, selective amount of albumin may pass the can pass through the glomerular filtration barrier (GFB) where it will be reabsorbed in the tubular compartment by the proximal tubule epithelial cells (PTEC). As Fetuin-A and albumin have similar properties, equivalent handling by the kidney can be expected. Fetuin-A can be absorbed in the proximal tubule in healthy rat by the megalin-mediated endocytosis, predominantly at the S1 segment and to the lower extent at the S2 segment. In condition where there is an accumulation of injury in the glomerulus, GFB will be disrupted and excessive amounts of different types of plasma proteins will leaked to the tubular compartment. This causes the amount of filtered protein needed to be reabsorbed by the proximal tubules to increase. As the reabsorption capacity of PTEC is not unlimited, protein from the circulation will present in the urine once this mechanism is saturated. Next to that, the filtered proteins may cause injury in the proximal tubules via various mechanisms, i.e. by obstructing tubular lumen with protein casts, by causing energy depletion and lysosomal rupture due to abundant amount of protein overload that needs to be reabsorbed by the PTEC, by triggering PTEC to release pro-inflammatory and pro-fibrotic cytokines, by causing intracellular accumulation of lipidated proteins that will trigger tubular apoptosis, and by activating complement system to cause direct injury to the PTEC. When this occurs, the ability of PTEC to locally produce Fetuin-A and to reabsorb filtered Fetuin-A can be impaired. As 24h uC-FetA excretion is significantly associated with an increased risk of graft function deterioration even after adjustment for potential confounders including 24h urinary protein excretion, the origin of uC-FetA can be from the local production by the PTEC instead of from the impaired reabsorption process. In the sensitivity analyses, uC-FetA may not be associated with graft function deterioration in patients with eGFR below 30 ml / min / 1.73m2. Fetuin-A from the circulation may also end up in the urine when there is an accumulated injury in the kidney. Thus, uC-FetA measured in this subgroup of patients may consists of locally produced Fetuin-A and circulating Fetuin-A. Fetuin- A in the circulation is not associated with kidney function.Attorney Docket No.132417-0002WO01

[0168] Both acute and chronic kidney injury are tightly associated with the occurrence of hypoxia. Among all structures within the kidney organ, PTEC are the most sensitive and most vulnerable, as these cells are highly active and have high oxygen demand. Because hypoxia plays a major role in the progression of the kidney disease and may present in the relatively early stages of kidney injury even before the structural injury occurred, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion-induced AKI, urinary Fetuin- A excretion increased before the surge of serum creatinine. Moreover, Fetuin-A deposition in the PTEC increased before structural injury in the tubules present morphologically. In type 2 diabetes mellitus patients, urinary Fetuin-A able to display progression of kidney function deterioration earlier than albuminuria. As one of the most important requirements for a biomarker is to reflect an underlying pathophysiology of the disease, in some embodiments, measurement of Fetuin-A in the urine may offer additional benefit as a biomarker over currently used parameters for early detection of graft injury in KTR such as serum creatinine or proteinuria.

[0169] ASSAY

[0170] In some embodiments, various methods, reagents, devices, and kits can be used to measure the level of Fetuin-A or a Fetuin-A-based fragment in various mediums, such as in a urine, serum, and other bodily fluids. In some embodiments, the enzyme-linked immunosorbent assay (ELISA) can be used to measure the level of Fetuin-A. For example, the ELISA can be used for quantitative measurement of Fetuin-A with specific post translational modification (PTM) in human urine and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists. For example, calibrators or unknown urine samples are mixed with an antibody having a binding affinity to a part of Fetuin-A, such as anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate pre-bounded with Fetuin-A or its segment, such as unique PTM Fetuin-A. The monoclonal antibody recognizes Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, in some embodiments, signal-generating molecule such as signal-molecule-tagged antibody can be used. In some embodiments, a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3',5,5'- tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve. FIRST NON-LIMITING EMBODIMENT SET

[0171] The present disclosure is also described by way of the following first non-limiting embodiment set. However, the use of these and other embodiments anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure. Likewise, the disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed,Attorney Docket No.132417-0002WO01 modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. 1. A method of preparing an assay, comprising: providing a first solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine fragment, to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a graft function deterioration in the recipient, wherein the first solution and the second solution are the same solution or different solution from each other. 2. The method of embodiment 1, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration. 3. The method of any one of preceeding embodiments, wherein the higher likelihood is higher than a likelihood of the graft function deterioration over the period of about ten years or less in a comparison recipient of a kidney transplantation having a ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg. 4. The method of any one of preceeding embodiments, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis. 5. The method of any one of preceeding embodiments, wherein the graft function deterioration is indicated by a decline in estimated Glomerular Filtration Rate (eGFR). 6. The method of any one of preceeding embodiments, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 7. The method of any one of preceeding embodiments, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 8. The method of any one of preceeding embodiments, wherein at least one reagent among the first reagent and the second reagent includes an antibody.Attorney Docket No.132417-0002WO01 9. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg. 10. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 25.5 ng / mg. 11. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 26 ng / mg. 12. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 27 ng / mg. 13. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 27.5 ng / mg. 14. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 28 ng / mg. 15. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 28.5 ng / mg. 16. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 28.7 ng / mg. 17. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 28.8 ng / mg. 18. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 28.9 ng / mg. 19. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 29.0 ng / mg. 20. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 29.1 ng / mg. 21. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 29.2 ng / mg. 22. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 29.3 ng / mg. 23. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 30 ng / mg. 24. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 34 ng / mg. 25. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 35 ng / mg. 26. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 37 ng / mg.Attorney Docket No.132417-0002WO01 27. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 38 ng / mg. 28. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 40 ng / mg. 29. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 50 ng / mg. 30. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 60 ng / mg. 31. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 65 ng / mg. 32. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 70 ng / mg. 33. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 71 ng / mg. 34. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 72 ng / mg. 35. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 73 ng / mg. 36. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 75 ng / mg. 37. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 80 ng / mg. 38. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 85 ng / mg. 39. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 90 ng / mg. 40. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 91 ng / mg. 41. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 92 ng / mg. 42. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 93 ng / mg. 43. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 94 ng / mg. 44. The method of any one of preceeding embodiments, wherein the ratio higher than about ng / mg is higher than about 90 ng / mg.Attorney Docket No.132417-0002WO01 45. The method of any one of preceeding embodiments, wherein the ratio higher than about 20 ng / mg is higher than about 95 ng / mg. 46. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 13 ng / mg. 47. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 12 ng / mg. 48. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 11 ng / mg. 49. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 10 ng / mg. 50. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 9 ng / mg. 51. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.8 ng / mg. 52. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.5 ng / mg. 53. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.4 ng / mg. 54. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.2 ng / mg. 55. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 8 ng / mg. 56. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.5 ng / mg. 57. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.4 ng / mg. 58. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.3 ng / mg. 59. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.3 ng / mg. 60. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.2 ng / mg. 61. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.1 ng / mg. 62. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 7 ng / mg.Attorney Docket No.132417-0002WO01 63. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 6 ng / mg. 64. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 5 ng / mg. 65. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 4 ng / mg. 66. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.9 ng / mg. 67. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.8 ng / mg. 68. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.7 ng / mg. 69. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.6 ng / mg. 70. The method of any one of preceeding embodiments, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.5 ng / mg. 71. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 5 % or higher. 72. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 8 % or higher. 73. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 10 % or higher. 74. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 15 % or higher. 75. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 16 % or higher. 76. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 20 % or higher. 77. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 22 % or higher. 78. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 25 % or higher. 79. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 30 % or higher. 80. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 32 % or higher.Attorney Docket No.132417-0002WO01 81. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 35 % or higher. 82. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 36 % or higher. 83. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 40 % or higher. 84. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 45 % or higher. 85. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 50 % or higher. 86. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 5 % to about 50%. 87. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 10 % to about 45%. 88. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 20 % to about 40%. 89. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 25 % to about 35%. 90. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 25 % to about 30%. 91. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 10 % to about 20%. 92. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 15 % to about 25%. 93. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 20 % to about 30%. 94. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 30 % to about 40%. 95. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 35 % to about 45%. 96. The method of any one of preceeding embodiments, wherein the higher likelihood of the graft function deterioration is about 40 % to about 50%. 97. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 2 years. 98. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 3 years.Attorney Docket No.132417-0002WO01 99. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 4 years. 100. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 5 years. 101. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 6 years. 102. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 7 years. 103. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 8 years. 104. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs after 9 years. 105. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs within 7 years. 106. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs within 8 years. 107. The method of any one of preceeding embodiments, wherein the graft function deterioration occurs within 9 years. 108. The method of any one of preceeding embodiments, further comprising assessing the probability of the graft function deterioration, based on the ratio and at least one other marker. 109. The method of embodiment 108, wherein the at least one other marker comprises age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof. 110. The method of embodiment 109, wherein the assessed probability exhibits at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). 111. The method of embodiment 110, wherein the assessed probability exhibits at least about 80 percent (%) of the AUC. 112. The method of embodiment 110, wherein the assessed probability exhibits at least about 85 percent (%) of the AUC 113. The method of embodiment 110, wherein the assessed probability exhibits at least about 90 percent (%) of the AUC 114. The method of embodiment 110, wherein the assessed probability exhibits at least about 95 percent (%) of the AUC. 115. An assay kit to determine a likelihood of a progressive renal decline, comprising:Attorney Docket No.132417-0002WO01 a first solution including a first reagent to interact with a post-translationally modified Fetuin- A fragments in urine fragment to indicate the level of the Fetuin A fragment in a urine sample from a recipient of a kidney transplantation after the kidney transplantation; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of the graft function deterioration over a period of about ten years or less, and wherein the first solution and the second solution are the same solution or different solution from each other. 116. The assay kit of embodiment 115, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration. 117. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood is higher than a likelihood of the graft function deterioration over the period of about ten years or less in a a comparison recipient of a kidney transplantation having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg. 118. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re- transplantation, or a need of dialysis. 119. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration is indicated by a decline in estimated Glomerular Filtration Rate (eGFR). 120. The assay kit of any one of preceeding embodiments after 115, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 121. The assay kit of any one of preceeding embodiments after 115, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 122. The assay kit of any one of preceeding embodiments after 115, wherein at least one reagent among the first reagent and the second reagent includes an antibody.Attorney Docket No.132417-0002WO01 123. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg. 124. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 25.5 ng / mg. 125. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 26 ng / mg. 126. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 27 ng / mg. 127. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 27.5 ng / mg. 128. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 28 ng / mg. 129. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 28.5 ng / mg. 130. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 28.7 ng / mg. 131. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 28.8 ng / mg. 132. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 28.9 ng / mg. 133. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 29.0 ng / mg. 134. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 29.1 ng / mg. 135. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 29.2 ng / mg. 136. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 29.3 ng / mg. 137. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 30 ng / mg. 138. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 34 ng / mg. 139. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 35 ng / mg. 140. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 37 ng / mg.Attorney Docket No.132417-0002WO01 141. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 38 ng / mg. 142. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 40 ng / mg. 143. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 50 ng / mg. 144. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 60 ng / mg. 145. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 65 ng / mg. 146. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 70 ng / mg. 147. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 71 ng / mg. 148. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 72 ng / mg. 149. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 73 ng / mg. 150. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 75 ng / mg. 151. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 80 ng / mg. 152. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 85 ng / mg. 153. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg. 154. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 91 ng / mg. 155. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 92 ng / mg. 156. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 93 ng / mg. 157. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 94 ng / mg. 158. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg.Attorney Docket No.132417-0002WO01 159. The assay kit of any one of preceeding embodiments after 115, wherein the ratio higher than about 20 ng / mg is higher than about 95 ng / mg. 160. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 13 ng / mg. 161. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 12 ng / mg. 162. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 11 ng / mg. 163. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 10 ng / mg. 164. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 9 ng / mg. 165. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.8 ng / mg. 166. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.5 ng / mg. 167. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.4 ng / mg. 168. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.2 ng / mg. 169. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 8 ng / mg. 170. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.5 ng / mg. 171. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.4 ng / mg. 172. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.3 ng / mg. 173. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.3 ng / mg. 174. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.2 ng / mg. 175. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.1 ng / mg. 176. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 7 ng / mg.Attorney Docket No.132417-0002WO01 177. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 6 ng / mg. 178. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 5 ng / mg. 179. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 4 ng / mg. 180. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.9 ng / mg. 181. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.8 ng / mg. 182. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.7 ng / mg. 183. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.6 ng / mg. 184. The assay kit of any one of preceeding embodiments after 115, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.5 ng / mg. 185. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 5 % or higher. 186. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 8 % or higher. 187. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 10 % or higher. 188. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 15 % or higher. 189. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 16 % or higher. 190. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 20 % or higher. 191. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 22 % or higher. 192. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 25 % or higher. 193. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 30 % or higher. 194. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 32 % or higher.Attorney Docket No.132417-0002WO01 195. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 35 % or higher. 196. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 36 % or higher. 197. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 40 % or higher. 198. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 45 % or higher. 199. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 50 % or higher. 200. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 5 % to about 50%. 201. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 10 % to about 45%. 202. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 20 % to about 40%. 203. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 25 % to about 35%. 204. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 25 % to about 30%. 205. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 10 % to about 20%. 206. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 15 % to about 25%. 207. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 20 % to about 30%. 208. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 30 % to about 40%. 209. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 35 % to about 45%. 210. The assay kit of any one of preceeding embodiments after 115, wherein the higher likelihood of the graft function deterioration is about 40 % to about 50%. 211. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 2 years. 212. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 3 years.Attorney Docket No.132417-0002WO01 213. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 4 years. 214. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 5 years. 215. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 6 years. 216. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 7 years. 217. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 8 years. 218. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs after 9 years. 219. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs within 7 years. 220. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs within 8 years. 221. The assay kit of any one of preceeding embodiments after 115, wherein the graft function deterioration occurs within 9 years. 222. The assay kit of any one of preceeding embodiments after 115, wherein the device is to determine the level of the Fetuin A fragment and the level of the urinary creatinine, to assess the probability of the graft function deterioration, based on the ratio and at least one other marker. 223. The assay kit of embodiment 222, wherein the at least one other marker comprises age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof. 224. The assay kit of embodiment 223, wherein the assessed probability exhibits at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). 225. The assay kit of embodiment 224, wherein the assessed probability exhibits at least about 80 percent (%) of the AUC. 226. The assay kit of embodiment 224, wherein the assessed probability exhibits at least about 85 percent (%) of the AUC. 227. The assay kit of embodiment 224, wherein the assessed probability exhibits at least about 90 percent (%) of the AUC. 228. The assay kit of embodiment 224, wherein the assessed probability exhibits at least about 95 percent (%) of the AUC. 229. A method of correlating a likelihood of a graft function deterioration, comprising:Attorney Docket No.132417-0002WO01 determining the level of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplantation after the kidney transplantation, wherein the first biomarker is a fragment of a post-translationally modified Fetuin-A fragments in urine fragment, wherein the second biomarker is a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a likelihood of a graft function deterioration in the recipient over a period of about ten years, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of the graft function deterioration over the period of about ten years or less. 230. The method of embodiment 229, wherein the ratio being higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to obviate the graft function deterioration. 231. The method of any one of preceeding embodiments after 229, wherein the higher likelihood is higher than a lower likelihood of the graft function deterioration over the period of about ten years or less in a comparison recipient of a kidney transplantation having a ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg. 232. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis. 233. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration is indicated by a decline in estimated Glomerular Filtration Rate (eGFR). 234. The method of any one of preceeding embodiments after 229, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 235. The method of any one of preceeding embodiments after 229, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 236. The method of any one of preceeding embodiments after 229, wherein at least one reagent among the first reagent and the second reagent includes an antibody. 237. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg. 238. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 25.5 ng / mg.Attorney Docket No.132417-0002WO01 239. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 26 ng / mg. 240. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 27 ng / mg. 241. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 27.5 ng / mg. 242. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 28 ng / mg. 243. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 28.5 ng / mg. 244. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 28.7 ng / mg. 245. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 28.8 ng / mg. 246. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 28.9 ng / mg. 247. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 29.0 ng / mg. 248. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 29.1 ng / mg. 249. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 29.2 ng / mg. 250. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 29.3 ng / mg. 251. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 30 ng / mg. 252. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 34 ng / mg. 253. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 35 ng / mg. 254. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 37 ng / mg. 255. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 38 ng / mg. 256. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 40 ng / mg.Attorney Docket No.132417-0002WO01 257. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 50 ng / mg. 258. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 60 ng / mg. 259. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 65 ng / mg. 260. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 70 ng / mg. 261. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 71 ng / mg. 262. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 72 ng / mg. 263. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 73 ng / mg. 264. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 75 ng / mg. 265. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 80 ng / mg. 266. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 85 ng / mg. 267. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg. 268. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 91 ng / mg. 269. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 92 ng / mg. 270. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 93 ng / mg. 271. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 94 ng / mg. 272. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg. 273. The method of any one of preceeding embodiments after 229, wherein the ratio higher than about 20 ng / mg is higher than about 95 ng / mg. 274. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 13 ng / mg.Attorney Docket No.132417-0002WO01 275. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 12 ng / mg. 276. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 11 ng / mg. 277. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 10 ng / mg. 278. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 9 ng / mg. 279. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.8 ng / mg. 280. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.5 ng / mg. 281. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.4 ng / mg. 282. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 8.2 ng / mg. 283. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 8 ng / mg. 284. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.5 ng / mg. 285. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.4 ng / mg. 286. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.3 ng / mg. 287. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.3 ng / mg. 288. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.2 ng / mg. 289. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.1 ng / mg. 290. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 7 ng / mg. 291. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 6 ng / mg. 292. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 5 ng / mg.Attorney Docket No.132417-0002WO01 293. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 4 ng / mg. 294. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.9 ng / mg. 295. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.8 ng / mg. 296. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.7 ng / mg. 297. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.6 ng / mg. 298. The method of any one of preceeding embodiments after 229, wherein the second ratio being lower than about 14 ng / mg is lower than about 3.5 ng / mg. 299. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 5 % or higher. 300. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 8 % or higher. 301. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 10 % or higher. 302. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 15 % or higher. 303. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 16 % or higher. 304. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 20 % or higher. 305. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 22 % or higher. 306. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 25 % or higher. 307. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 30 % or higher. 308. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 32 % or higher. 309. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 35 % or higher. 310. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 36 % or higher.Attorney Docket No.132417-0002WO01 311. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 40 % or higher. 312. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 45 % or higher. 313. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 50 % or higher. 314. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 5 % to about 50%. 315. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 10 % to about 45%. 316. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 20 % to about 40%. 317. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 25 % to about 35%. 318. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 25 % to about 30%. 319. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 10 % to about 20%. 320. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 15 % to about 25%. 321. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 20 % to about 30%. 322. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 30 % to about 40%. 323. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 35 % to about 45%. 324. The method of any one of preceeding embodiments after 229, wherein the higher likelihood of the graft function deterioration is about 40 % to about 50%. 325. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 2 years. 326. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 3 years. 327. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 4 years. 328. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 5 years.Attorney Docket No.132417-0002WO01 329. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 6 years. 330. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 7 years. 331. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 8 years. 332. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs after 9 years. 333. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs within 7 years. 334. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs within 8 years. 335. The method of any one of preceeding embodiments after 229, wherein the graft function deterioration occurs within 9 years. 336. The method of any one of preceeding embodiments after 229, further comprising assessing the probability of the graft function deterioration, based on the ratio and at least one other marker. 337. The method of embodiment 336, wherein the at least one other marker comprises age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof. 338. The method of embodiment 337, wherein the assessed probability exhibits at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC). 339. The method of embodiment 338, wherein the assessed probability exhibits at least about 80 percent (%) of the AUC. 340. The method of embodiment 338, wherein the assessed probability exhibits at least about 85 percent (%) of the AUC. 341. The method of embodiment 338, wherein the assessed probability exhibits at least about 90 percent (%) of the AUC. 342. The method of embodiment 338, wherein the assessed probability exhibits at least about 95 percent (%) of the AUC. SECOND NON-LIMITING EMBODIMENT SET

[0172] The present disclosure is also described by way of the following second non-limiting embodiment set. However, the use of these and other embodiments anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure. Likewise, the disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed,Attorney Docket No.132417-0002WO01 modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. 1. A method of preparing an assay, comprising: providing a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less. 2. The method of embodiment 1, wherein the excretion being higher than about 0.8 µg / h triggers a medical intervention to suppress the graft function deterioration. 3. The method of any of embodiments 1-2, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis. 4. The method of any of embodiments 1-3, wherein the recipient has been indicated to have the eGFR of about 25 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher at a time around collecting the urine sample. 5. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 2 hours. 6. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 4 hours. 7. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 6 hours. 8. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 12 hours. 9. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 24 hours. 10. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 36 hours. 11. The method of any of embodiments 1-4, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 48 hours. 12. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 day after the kidney transplantation.Attorney Docket No.132417-0002WO01 13. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 days after the kidney transplantation. 14. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 week after the kidney transplantation. 15. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 weeks after the kidney transplantation. 16. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 month after the kidney transplantation. 17. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 3 months after the kidney transplantation. 18. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 6 months after the kidney transplantation. 19. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 12 months after the kidney transplantation. 20. The method of any of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 24 months after the kidney transplantation. 21. The method of any of embodiments 1-20, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about nine years or less. 22. The method of any of embodiments 1-20, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about seven years or less. 23. The method of any of embodiments 1-20, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about five years or less. 24. The method of any of embodiments 1-20, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about three years or less. 25. The method of any of embodiments 1-20, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about one year or less. 26. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 0.9 µg / h. 27. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 1 µg / h.Attorney Docket No.132417-0002WO01 28. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2 µg / h. 29. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.1 µg / h. 30. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.2 µg / h. 31. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.3 µg / h. 32. The method of any of embodiments 1-25, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.5 µg / h. 33. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 34. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.5 or more. 35. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.59 or more. 36. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.6 or more. 37. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.7 or more. 38. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.78 or more. 39. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.79 or more. 40. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.8 or more.Attorney Docket No.132417-0002WO01 41. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.1 or more. 42. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.15 or more. 43. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.18 or more. 44. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 45. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.22 or more. 46. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.5 to about 2.5. 47. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.6 to about 2.3. 48. The method of any of embodiments 1-32, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.59 to about 2.22. 49. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 50. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 51. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.26 or more. 52. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.5 or more.Attorney Docket No.132417-0002WO01 53. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.7 or more. 54. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3 or more. 55. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.5 or more. 56. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.6 or more. 57. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more. 58. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more. 59. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4 or more. 60. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.5 or more. 61. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.7 or more. 62. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.8 or more. 63. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.9 or more. 64. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 5 or more.Attorney Docket No.132417-0002WO01 65. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5. 66. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5. 67. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2.5 to about 5. 68. The method of any of embodiments 1-48, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 3.5 to about 5. 69. The method of any of embodiments 1-68, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A. 70. The method of any of embodiments 1-68, wherein the reagent includes an antibody. 71. An assay kit to determine a likelihood of a graft function deterioration of a transplanted kidney, comprising: a solution including a concentration of a reagent to interact with an amount of a urinary connecting peptide-containing Fetuin A fragment in a urine sample from a recipient of a kidney transplantation; a device to measure an average Fetuin A fragment excretion to urine from the solution, to correlate the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less. 72. The assay kit of embodiment 71, wherein the excretion being higher than about 0.8 µg / h triggers a medical intervention to suppress the graft function deterioration. 73. The assay kit of any of embodiments 71-72, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis. 74. The assay kit of any of embodiments 71-73, wherein the recipient has been indicated to have the eGFR of about 25 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher at a time around collecting the urine sample. 75. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 2 hours. 76. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 4 hours.Attorney Docket No.132417-0002WO01 77. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 6 hours. 78. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 12 hours. 79. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 24 hours. 80. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 36 hours. 81. The assay kit of any of embodiments 71-74, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 48 hours. 82. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 day after the kidney transplantation. 83. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 days after the kidney transplantation. 84. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 week after the kidney transplantation. 85. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 weeks after the kidney transplantation. 86. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 month after the kidney transplantation. 87. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 3 months after the kidney transplantation. 88. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 6 months after the kidney transplantation. 89. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 12 months after the kidney transplantation. 90. The assay kit of any of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 24 months after the kidney transplantation. 91. The assay kit of any of embodiments 71-90, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about nine years or less. 92. The assay kit of any of embodiments 71-90, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about seven years or less.Attorney Docket No.132417-0002WO01 93. The assay kit of any of embodiments 71-90, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about five years or less. 94. The assay kit of any of embodiments 71-90, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about three years or less. 95. The assay kit of any of embodiments 71-90, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about one year or less. 96. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 0.9 µg / h. 97. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 1 µg / h. 98. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2 µg / h. 99. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.1 µg / h. 100. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.2 µg / h. 101. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.3 µg / h. 102. The assay kit of any of embodiments 71-95, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.5 µg / h. 103. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 104. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.5 or more. 105. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.59 or more. 106. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.6 or more.Attorney Docket No.132417-0002WO01 107. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.7 or more. 108. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.78 or more. 109. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.79 or more. 110. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.8 or more. 111. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.1 or more. 112. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.15 or more. 113. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.18 or more. 114. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 115. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.22 or more. 116. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.5 to about 2.5. 117. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.6 to about 2.3.Attorney Docket No.132417-0002WO01 118. The assay kit of any of embodiments 71-102, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.59 to about 2.22. 119. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 120. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 121. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.26 or more. 122. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.5 or more. 123. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.7 or more. 124. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3 or more. 125. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.5 or more. 126. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.6 or more. 127. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more. 128. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more.Attorney Docket No.132417-0002WO01 129. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4 or more. 130. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.5 or more. 131. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.7 or more. 132. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.8 or more. 133. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.9 or more. 134. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 5 or more. 135. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5. 136. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5. 137. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2.5 to about 5. 138. The assay kit of any of embodiments 71-117, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 3.5 to about 5. 139. The assay kit of any of embodiments 71-138, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A. 140. The assay kit of any of embodiments 71-138, wherein the reagent includes an antibody. 141. A method of correlating a likelihood of a graft function deterioration in a recipient of a kidney transplantation, comprising:Attorney Docket No.132417-0002WO01 obtaining a solution including a urine sample from a recipient of a kidney transplantation after the kidney transplantation and a reagent to interact with a urinary connecting peptide-containing Fetuin A fragment; measuring an average Fetuin A fragment excretion to urine from the solution; correlating the measured average Fetuin A fragment excretion with a likelihood of a graft function deterioration in the recipient, wherein the measured average Fetuin A fragment excretion being higher than about 0.8 micrograms per hour (µg / h) indicates a higher likelihood of the graft function deterioration over a period of about ten years or less. 142. The method of embodiment 141, wherein the excretion being higher than about 0.8 µg / h triggers a medical intervention to suppress the graft function deterioration. 143. The method of any of embodiments 141-142, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis. 144. The method of any of embodiments 141-143, wherein the recipient has been indicated to have the eGFR of about 25 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher at a time around collecting the urine sample. 145. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 2 hours. 146. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 4 hours. 147. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 6 hours. 148. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 12 hours. 149. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 24 hours. 150. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 36 hours. 151. The method of any of embodiments 141-144, wherein the average Fetuin A fragment excretion to the urine is averaged over no less than about 48 hours. 152. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 day after the kidney transplantation. 153. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 days after the kidney transplantation. 154. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 week after the kidney transplantation.Attorney Docket No.132417-0002WO01 155. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 2 weeks after the kidney transplantation. 156. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 1 month after the kidney transplantation. 157. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 3 months after the kidney transplantation. 158. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 6 months after the kidney transplantation. 159. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 12 months after the kidney transplantation. 160. The method of any of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplantation at least about 24 months after the kidney transplantation. 161. The method of any of embodiments 141-160, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about nine years or less. 162. The method of any of embodiments 141-160, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about seven years or less. 163. The method of any of embodiments 141-160, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about five years or less. 164. The method of any of embodiments 141-160, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about three years or less. 165. The method of any of embodiments 141-160, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h indicates a higher likelihood of the graft function deterioration of the transplanted kidney over a period of about one year or less. 166. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 0.9 µg / h. 167. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 1 µg / h. 168. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2 µg / h. 169. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.1 µg / h.Attorney Docket No.132417-0002WO01 170. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.2 µg / h. 171. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.3 µg / h. 172. The method of any of embodiments 141-165, wherein the average Fetuin A fragment excretion being higher than about 0.8 µg / h is higher than about 2.5 µg / h. 173. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 174. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.5 or more. 175. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.59 or more. 176. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.6 or more. 177. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.7 or more. 178. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.78 or more. 179. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.79 or more. 180. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 1.8 or more. 181. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.1 or more. 182. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.15 or more.Attorney Docket No.132417-0002WO01 183. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.18 or more. 184. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 185. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.22 or more. 186. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.5 to about 2.5. 187. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.6 to about 2.3. 188. The method of any of embodiments 141-172, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being from about 0.9 µg / h to about 2.4 µg / h and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 1.59 to about 2.22. 189. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2 or more. 190. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.2 or more. 191. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.26 or more. 192. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.5 or more. 193. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 2.7 or more.Attorney Docket No.132417-0002WO01 194. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3 or more. 195. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.5 or more. 196. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.6 or more. 197. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more. 198. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 3.7 or more. 199. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4 or more. 200. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.5 or more. 201. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.7 or more. 202. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.8 or more. 203. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 4.9 or more. 204. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is about 5 or more. 205. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5.Attorney Docket No.132417-0002WO01 206. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2 to about 5. 207. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 2.5 to about 5. 208. The method of any of embodiments 141-188, wherein a hazard ratio corresponding to the average Fetuin A fragment excretion being about 2.4 µg / h or higher and corresponding to the average Fetuin A fragment excretion being about 0.9 µg / h or lower is from about 3.5 to about 5. 209. The method of any of embodiments 141-208, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A. 210. The method of any of embodiments 141-208, wherein the reagent includes an antibody. NON-LIMITING EXAMPLES

[0173] The following examples are provided to illustrate selected embodiments. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. Thus, the examples provided below, while illustrated with a particular medical device or active agent, are applicable to the range of medical devices and active agents described herein. Example 1-1

[0174] The objective of the clinical performance study illustrated in Examples 1-5 is to evaluate the prognostic performance of the Human uPTM3-DKD ELISA test of graft failure for kidney transplantation receivers (KTR).

[0175] Design and Study Population

[0176] We used data from TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841)2. All adult KTRs visiting the University Medical Center Groningen (UMCG) outpatient clinic between November 2008 and March 2011 with a functioning graft for at least 1 year after transplantation, without any drug or alcohol addiction or systemic illnesses, were invited to participate in the cohort. During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participants and gave written informed consent. Example 1-2

[0177] Parameters of clinical performance determined

[0178] Hazard ratios and 95% confidence intervals calculated based on Cox proportional hazards regression analysis will be use to evaluate the ability of uPTM-FetA in assessing the risk of kidney graft failure defined as the need of re-transplantation or re-initiation of dialysis.

[0179] Statistics & Sample Size Calculation

[0180] The required sample size was estimated based on the following assumptions:Attorney Docket No.132417-0002WO01 1. The hazard ratio (θ) of the high-risk group relative to the low-risk group is expected to be a value greater than 1, and this study sets a hazard ratio of 2.5; 2. According to literature statistics, about 20% of KTRs will develop graft function decline6. Therefore, this study estimated that the incidence rate was 20% (pE= 0.20); 3. The proportion of subjects in the low-risk group and the high-risk group is the 3:1 (pL=0.75; pH=0.25); 4. Type I error(α) is 0.05; 5. power (1-β) is 80%;

[0181] The number of samples is calculated as7 ((z1-α / 2+z1-β) / (lnθ-ln(1)))2 / (pLpHpE). Under the assumptions above, 249 patients are required. Example 1-3

[0182] Data and results management

[0183] The excel files containing a series of clinical records of the KTRs in TransplantLines Food and Nutrition Biobank and Cohort Study will be manually downloaded and saved by the Principal Investigator, in a specific folder on a computer with an encrypted disk and other privacy and safety measures, in alignment with GDPR. The folder would only be accessible to the PI and the Statistician. More details regarding the data storage, back-up, selection and preservation are available below. The urine samples will be transferred to BPM and then be stored at -80°C in accordance with the sample management procedure of BPM, and will not be taken out until it is used. After testing, test results will be recorded in excel files and saved by the BPM testing laboratory personnel, in a specific folder as previously described.

[0184] Data and results analysis

[0185] This study appears to be focused on determining the optimal threshold for differentiating high-risk and low-risk subjects for kidney graft failure in KTRs using the uPTM-FetA / UCr. The primary endpoint of this study was death-censored graft failure defined as the need of re- transplantation or re-initiation of dialysis. The study design involves dividing the KTRs with spot urine sample into two subsets: the Primary Set, which will consist of 50% of the KTRs and will be used to determine the clinical cut-off using the Youden Index8; and the Validation Set, which will consist of the remaining 50% of the KTRs and will be used to validate the cut-off.

[0186] In routine practice of urinary test, spot urine is preferred to a 24-hour urine sample particularly if 24 hours urine sample is difficult to obtain or unavailable. However, 24-hour urinalysis is still generally used in the metabolic evaluation of several types of kidney disorders. In this study, to validate the generalness of prognosis ability of the kit for different type of urine samples, we define the KTRs with uPTM-FetA and urine creatinine measured from 24h urine sample as "24h Set", and conduct a cut-off validation analysis.Attorney Docket No.132417-0002WO01

[0187] After determining the cut-off in the Primary Set, KTRs will be divided into two groups based on their uPTM-FetA / UCr levels: low-risk (uPTM-FetA / UCr < cut-off) and high-risk (uPTM- FetA / UCr ≥ cut-off). The Cox proportional hazard model and Kaplan-Meier graph / log-rank test will be used to assess the hazard ratio and compare the survival function of the two groups and the prognosis ability of uPTM-FetA / UCr in kidney graft failure will be assessed using the Cox proportional hazard model and Kaplan-Meier graph / log-rank test in both sets.

[0188] Proteinuria is commonly used as a prognostic marker after kidney transplantation9,10. It is generally accepted that one-year post-transplant proteinuria over 0.5 gm per day predicts the development of chronic allograft nephropathy and poor graft outcome11-13. To demonstrate the ability of uPTM-FetA / UCr in providing additional interpretive information in clinical use, we conduct comparative analyses to evaluate the event rate of kidney graft failure in comparison to uPTM- FetA / UCr (above and below the clinical cut-off) and proteinuria (above and below 0.5 g / day) by using Cox regression model upon adjustment for baseline risk factors of age and gender. Example 1-4

[0189] Results

[0190] Among the 707 KTRs, 173 and 72 patients without sufficient amount of spot urine and 24- hour urine, respectively, available for uPTM-FetA measurement. Therefore, we excluded these patients and 534 KTRs were used for analysis based on the spot urine and 635 KTRs were used for analysis based on 24-hour urine samples.

[0191] The 534 KTRs with spot urine samples were divided into two sets: The Primary Set includes 267(50%) KTRs and the Validation Set includes 267(50%) KTRs. The 635 KTRs with uPTM-FetA and urine creatinine measured from 24-hour urine sample was defined as 24h Set.

[0192] Results of Primary Set

[0193] The mean age of the 267 KTRs was 53.93 years, 116 (43%) were female, eGFR was 51.69 ml / min / 1.73m2, and the median baseline Protein excretion was 0.21 g / d. Among 267 KTRs, 35 (13%) experienced a kidney graft failure during follow-up, and their median survival time was 6.04 years (IQR, 4.62-6.40 years). The clinical cut-off of uPTM-FetA / UCr obtained by Youden Index method was 29.1 ng / mg (equivalent to IVD103 cut-off 1.464). According to this clinical cut-off, subjects were divided into low-risk group (uPTM-FetA / UCr<29.1 ng / mg) and high-risk group (uPTM- FetA / UCr≥29.1 ng / mg) (Table 1). From Table 1, there was a significant difference in the frequency of kidney graft failure between the low-risk group and the high-risk group (8.1% and 31%, p- value<0.001) and the survival time is significantly shorter for high-risk group than that of low-risk (4.64[2.82, 6.15] vs.6.08[5.65, 6.46], p-value<0.001).Attorney Docket No.132417-0002WO01 Table 1. Summary of 267 KTRs in Primary Set Risk groupVariableOverall,Low risk, High risk, N = 2671N = 209 N = 58 p-value2Age, years, 53.93±12.65 53.41±12.69 55.81±12.41 0.198 Gender (F), n (%), 116 (43%) 89 (43%) 27 (47%) 0.697 BMI, kg / m², 26.43±4.49 26.26±4.25 27.06±5.27 0.288 eGFR, ml / min / 1.73m², 51.69±19.58 54.43±18.46 41.83±20.47 <0.001 Protein concentration, g / L, 0.10 [0.00, 0.20] 0.10 [0.00, 0.10] 0.25 [0.10, 0.70] <0.001 Protein excretion, g, 0.21 [0.00, 0.40] 0.18 [0.00, 0.27] 0.54 [0.28, 1.50] <0.001 proteinuria, n (%), 57 (21%) 28 (13%) 29 (50%) <0.001 Time since transplantation, years, 5.29 [2.63, 11.39] 5.26 [2.84, 11.36] 5.55 [1.66, 11.72] 0.930 Primary kidney disease, n (%), 0.319 Unknown, 40 (15%) 34 (16%) 6 (10%) Glomerulonephritis, 75 (28%) 60 (29%) 15 (26%) Interstitial-nephritis, 32 (12%) 26 (12%) 6 (10%) Cystic kidney disease, 62 (23%) 48 (23%) 14 (24%) Other congenital and hereditary kidney disease, 12 (4.5%) 8 (3.8%) 4 (6.9%) Renal vascular disease, excluding vasculitis, 11 (4.1%) 7 (3.3%) 4 (6.9%) Diabetes Mellitus, 13 (4.9%) 7 (3.3%) 6 (10%) Other multisystem diseases, 17 (6.4%) 15 (7.2%) 2 (3.4%) Other, 5 (1.9%) 4 (1.9%) 1 (1.7%) Acute rejection, n (%), 76 (28%) 56 (27%) 20 (34%) 0.325 HLA class I antibodies positive, n (%), 40 (15%) 31 (15%) 9 (16%) >0.999 HLA class II antibodies positive, n (%), 46 (17%) 33 (16%) 13 (22%) 0.324 Kidney donor status, n (%), 0.958 Deceased, 178 (67%) 140 (67%) 38 (66%) Living, 89 (33%) 69 (33%) 20 (34%) Donor age, years, 43.20±15.00 42.87±14.53 44.39±16.68 0.537 SBP, mmHg, 135.92±17.80 134.76±16.60 140.16±21.24 0.080 DBP, mmHg, 81.67±11.05 81.25±10.06 83.23±14.08 0.323 Diabetes mellitus, n(%), 55 (21%) 42 (20%) 13 (22%) 0.839 Plasma glucose, mg / dl, 5.10 [4.70, 5.80] 5.10 [4.70, 5.60] 5.20 [4.62, 5.90] 0.750 HbA1c, %, 5.91±0.65 5.90±0.64 5.93±0.68 0.747 uPTM-FetA, ng / mL, 7.76 [3.99, 17.48] 5.98 [2.97, 11.21] 33.34 [19.49, 65.08] <0.001 uPTM-FetA / Cr, ng / mg, 10.49 [4.74, 26.79] 7.34 [3.89, 13.99] 54.43 [34.91, 93.63] <0.001 Total_uPTM-FetA, μg, 36.77 [18.50, 67.19] 28.32 [16.78, 52.39] 114.32 [57.71, 372.85] <0.001 IVD103 1.00±0.68 0.77±0.56 1.82±0.39 <0.001 Graft Failure, n (%), 35 (13%) 17 (8.1%) 18 (31%) <0.001 Survival Time, year, 6.04 [4.62, 6.40] 6.08 [5.65, 6.46] 4.64 [2.82, 6.15] <0.0011Mean±SD; n (%); Median [IQR]2Welch Two Sample t-test; Pearson's Chi-squared test; Kruskal-Wallis rank sum testAttorney Docket No.132417-0002WO01 Table 2. Cumulative probability (with 95% confidence intervals) of event for kidney graft failure at various time points for KTRs in the Primary Set. Time points Category 2 Year 3 Year 4 Year 5 Year 1.5%4.0% 6.1% 6.7%uPTM-FetA / UCr<29.1 ng / mg(0% to 3.1%)(1.2% to 6.7%) (2.7% to 9.4%) (3.1% to 10%) 8.8%16% 25% 36%uPTM-FetA / UCr≥29.1 ng / mg(1.1% to 16%)(6.0% to 26%) (12% to 36%) (21% to 49%) Table 3. Cox Proportional Hazards Regression Models of Primary Set

[0194] Notation: The reference category for uPTM-FetA / UCr is the low-level uPTM- FetA / UCr<29.1 ng / mg; The reference group for gender is male; Age and eGFR are included as continuous variables.

[0195] Figure.6 illustrates a Kaplan-Meier Survival Curve for KTRs with death-censored graft failure of Primary Set in some embodiments. Referring to Figure 1, The Kaplan-Meier estimate indicated that the KTRs in high-risk group had a significant risk of events relative to the KTRs in low- risk group, with a log-rank test p-value of <0.0001. The cumulative probabilities for events in the Primary Set by baseline uPTM-FetA / UCr risk category at time points of 2-5 years after baseline were also displayed in Table 2.

[0196] Further investigation with univariate Cox proportional hazards model indicated that the high- risk group had a significant risk of graft failure than the low-risk group, with a hazard ratio of 4.98 (95% CI: 2.56 – 9.69, p-value<0.001). Thus, uPTM-FetA / UCr was a significant predictor of enhance risk of kidney graft failure (Table 3). Multivariate Cox proportional hazards model adjusted for age, sex, and eGFR was also showed in Table 3. The uPTM-FetA / UCr was attenuated from the univariate analysis but remained a highly significant and independent predictor of risk for kidney graft failure with a hazard ratio of 2.88 (95% CI: 1.42 – 5.84, p-value=0.003). The reduced hazard probably reflects that uPTM-FetA / UCr may be related to the clinical factors but is clearly providing additional information.Attorney Docket No.132417-0002WO01

[0197] In addition, a comparative analysis of uPTM-FetA / UCr, proteinuria and event rate using the urine samples from the Primary Set was provided to present additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 4). The analysis evaluated the event rate for the primary endpoint in comparison to the uPTM-FetA / UCr (above and below the cut-off 29.1 ng / mg) and Proteinuria (total protein excretion above and below 0.5 g / day). Comparing to the reference group of uPTM-FetA / UCr<29.1 ng / mg and non-Proteinuria, hazard ratios are 3.01 (95% CI: 0.79-11.5, p- value=0.107), 8.82 (95% CI: 3.39-22.9, p-value<0.001) and 18.5 (95% CI: 7.77-44.2, p-value<0.001) for the second, third and fourth category, respectively.

[0198] Figure 2A illustrates a Kaplan-Meier curve for KTRs in the Primary Set with non- Proteinuria, according to a non-limiting embodiment. Figure 2B illustrates a Kaplan-Meier curve for KTRs in the Primary Set with Proteinuria by uPTM-FetA / UCr risk category, according to a non- limiting embodiment. Referring to Figures 2A and 2B, the Kaplan-Meier curves in Figures 2A and 7 BN showed that the KTRs with uPTM-FetA / UCr≥29.1 ng / mg had a slightly significant risk of events relative to the low uPTM-FetA / UCr group for both non-Proteinuria and Proteinuria with log-rank test p-values 0.12 and 0.074, respectively. This suggested that uPTM-FetA / UCr can provide additional information to Proteinuria in assessing risk of kidney graft failure. Table 4. Hazard ratios of kidney graft failure by uPTM-FetA / UCr and Proteinuria category for 267 KTRs in the Primary Set. Category*N Number ofHR (95% CI)1 p-events value1. uPTM-FetA / UCr<29.1 ng / mg & non- Reference --- Proteinuria181 82. uPTM-FetA / UCr≥29.1 ng / mg & non- 3.01 (0.79 to Proteinuria29 311.5)0.1073. uPTM-FetA / UCr<29.1 ng / mg & Proteinuria28 98.82 (3.39 to<0.00122.9)4. uPTM-FetA / UCr≥29.1 ng / mg & Proteinuria29 1518.5 (7.77 to<0.00144.2)1HR = Hazard Ratio, CI = Confidence Interval *Adjusted for age and sex

[0199] Results of Validation Set

[0200] The mean age of the 267 KTRs was 52.32 years, 111 (42%) were female, the mean baseline eGFR was 51.95 ml / min / 1.73m2, and the median baseline Protein excretion was 0.18 g / d. Among 267 KTRs, 29(11%) experienced a kidney graft failure during follow-up, and their median survival time was 5.06 years (IQR: 4.47 - 5.36 years). According to the clinical cut-off 29.1 ng / mg derived from Primary Set, KTRs were divided into low-risk group (uPTM-FetA / UCr<29.1 ng / mg) and high-riskAttorney Docket No.132417-0002WO01 group (uPTM-FetA / UCr≥29.1 ng / mg) (Table 5). From Table 5, there was a significant difference in the frequency of kidney graft failure between the low-risk group and the high-risk group (7.6% and 29%, p-value< 0.001) and the survival time is slightly significantly shorter for high-risk group than that of low-risk (4.98 [2.34, 5.34] vs 5.06 [4.55, 5.36], p-value=0.100). Table 5. of 267 KTRs in Validation Set Risk group Variable Overall, Low risk, High risk, N = 2671N = 225 p-value2Age, years, 52.32±13.36 51.93±13.420.258 Gender (F), n (%), 111 (42%) 92 (41%) 19 (45%) 0.723 BMI, kg / m², 26.71±4.84 26.57±4.89 27.44±4.60 0.271 eGFR, ml / min / 1.73m², 51.95±20.39 53.86±19.62 41.81±21.65 0.001 Protein concentration, g / L, 0.10 [0.00, 0.10] 0.10 [0.10, 0.48] <0.001 Protein excretion, g, 0.16 [0.00, 0.26] 0.29 [0.15, 0.88] <0.001 proteinuria, n (%), 28 (12%) 18 (43%) <0.001 Time since transplantation, years,4.99 [1.05, 10.96] 5.03 [1.05, 14.23] 0.606 Primary kidney disease, n (%), 0.362 Unknown, 45 (17%)8 (19%) Glomerulonephritis, 66 (25%) 59 (26%) 7 (17%) Interstitial-nephritis, 29 (11%) 24 (11%) 5 (12%) Cystic kidney disease, 53 (20%) 42 (19%) 11 (26%) Other congenital and hereditary kidney 12 (4.5%) 12 (5.3%) 0 (0%) disease, Renal vascular disease, excluding vasculitis, 16 (6.0%)4 (9.5%) Diabetes Mellitus, 14 (5.2%)2 (4.8%) Other multisystem diseases, 23 (8.6%)2 (4.8%) Other, 9 (3.4%)3 (7.1%) Acute rejection, n(%), 68 (25%)11 (26%) >0.999 HLA class I antibodies positive, n (%), 38 (14%)9 (21%) 0.225 HLA class II antibodies positive, n (%), 42 (16%)7 (17%) >0.999 Kidney donor status, n(%), 0.240 Deceased, 166 (62%) 136 (60%) 30 (71%) Living, 101 (38%) 89 (40%) 12 (29%) Donor age, years, 43.39±15.21 43.51±14.55 42.79±18.49 0.811 SBP, mmHg, 136.56±18.17 136.60±18.17 136.33±18.39 0.932 DBP, mmHg, 83.10±11.70 83.43±11.63 81.33±12.01 0.300 Diabetes mellitus, n (%), 66 (25%) 53 (24%) 13 (31%) 0.409 Plasma glucose, mg / dl, 5.30 [4.80, 6.00] 5.30 [4.80, 6.00] 5.60 [5.20, 6.80] 0.006 HbA1c, %, 6.04±0.93 6.02±0.83 6.09±1.36 0.756 uPTM-FetA, ng / mL, 7.07 [2.86, 14.82] 5.65 [2.16, 10.55] 21.46 [16.45, 38.41] <0.001 uPTM-FetA / UCr, ng / mg, 10.31 [4.03, 22.44] 8.46 [3.38, 15.08] 46.15 [37.14, 71.43] <0.001 IVD103, ng / mg, 0.96±0.57 0.81±0.50 1.73±0.22 <0.001 Graft Failure, n (%), 29 (11%) 17 (7.6%) 12 (29%) <0.001Attorney Docket No.132417-0002WO01

[0201] Figure 3 illustrates a Kaplan-Meier Survival Curve for KTRs with death-censored graft failure of Validation Set, according to a non-limiting embodiment. Referring to Figure 3, the Kaplan- Meier estimate indicated that the KTRs in high-risk group had a significant risk of events relative to the KTRs in low-risk group, with a log-rank test p value of <0.0001. The cumulative probabilities for events in the Validation Set by baseline uPTM-FetA / UCr risk category at time points of 2-5 years after baseline were also displayed in Table 6. Table 6. Cumulative probability (with 95% confidence intervals) of event for kidney graft failure at various time points for subjects in the Validation Set. Time points Category 2 Year 3 Year 4 Year 5 Year uPTM-FetA / UCr<29.1 3.6% 5.5% 6.5% 8.3% ng / mg (1.1% to 6.1%) (2.4% to 8.5%) (3.2% to 9.8%) (4.4% to 12%) uPTM-FetA / UCr≥29.1 20% 22% 25% 32% ng / mg (6.6% to 31%) (8.4% to 34%) (10% to 38%) (15% to 45%) Table 7. Cox Proportional Hazards Regression Models of Validation Set(<29.1 ng / mg); The reference group for gender is male; Age and eGFR are included as continuous variables.Attorney Docket No.132417-0002WO01

[0202] Further investigation with univariate Cox proportional hazards model indicated that the high- risk group had a significant risk of graft failure than the low-risk group, with a hazard ratio of 4.51 (95% CI: 2.15 – 9.45, p-value<0.001). Thus, uPTM-FetA / UCr was a significant predictor of enhance risk of kidney graft failure (Table 7). Multivariate Cox proportional hazards model adjusted for age, sex, and eGFR was also showed in Table 7. The uPTM-FetA / UCr was attenuated from the univariate analysis but remained a significant and independent predictor of risk for kidney graft failure with a hazard ratio of 2.36 (95% CI: 1.09 – 5.15, p-value=0.030). The reduced hazard probably reflects that uPTM-FetA / UCr may be related to the clinical factors but is clearly providing additional information.

[0203] In addition, a comparative analysis of uPTM-FetA / UCr, proteinuria and event rate using the urine samples from the Primary Set was provided to present additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 8). The analysis evaluated the event rate for the primary endpoint in comparison to the uPTM-FetA / UCr (above and below the cut-off 29.1 ng / mg) and Proteinuria (total protein excretion above and below 0.5 g / d). Comparing to the reference group of uPTM-FetA / UCr <29.1 ng / mg and non-Proteinuria, hazard ratios are 2.93 (95% CI: 0.93-9.23, p- value=0.066), 3.46 (95% CI: 1.19-10.1, p-value=0.023) and 13.1 (95% CI: 5.08 – 33.5, p- value<0.001) for the second, third and fourth category, respectively.

[0204] Figure 4A illustrates a Kaplan-Meier curve for subjects in the Validation Set with non- Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment. Figure 4B illustrates a Kaplan-Meier curve for subjects in the Validation Set with Proteinuria by uPTM- FetA / UCr risk category, according to a non-limiting embodiment. Referring to Figures 4A and 4B, The Kaplan-Meier curves also showed that the patients with uPTM-FetA / UCr ≥ 29.1 ng / mg had a significant risk of events relative to the low uPTM-FetA / UCr group for both non-Proteinuria and Proteinuria with log-rank test p-values 0.061 and 0.015, respectively. This suggested that uPTM- FetA / UCr can provide additional information to Proteinuria in assessing risk of kidney graft failure.

[0205] Analyses by Validation Set provided positive validation, confirming that the utility of the clinical cut-off for uPTM-FetA / UCr has good predictive power for KTRs at risk of graft failure. Table 8. Hazard ratios of kidney graft failure by uPTM-FetA / UCr and Proteinuria category for 267 KTRs in the Validation Set. Category* N Number of events HR (95% CI)1p-value 1. uPTM-FetA / UCr<29.1 ng / mg & non- Reference --- Proteinuria197 122. uPTM-FetA / UCr≥29.1 ng / mg & non- Proteinuria24 4 2.93 (0.93 to 9.23) 0.0663. uPTM-FetA / UCr<29.1 ng / mg & Proteinuria28 5 3.46 (1.19 to 10.1) 0.0234. uPTM-FetA / UCr≥29.1 ng / mg & Proteinuria18 8 13.1 (5.08 to 33.5) <0.001Attorney Docket No.132417-0002WO01 Category* N Number of events HR (95% CI)1p-value 1HR = Hazard Ratio, CI = Confidence Interval *Adjusted for age and sex

[0206] Results of 24h Set

[0207] The mean age of the 635 KTRs was 53 years, 268 (42%) were female, the mean baseline eGFR was 52.31 ml / min / 1.73m2, and the median baseline Protein excretion was 0.20 g / d. Among 635 KTRs, 75(12%) experienced a kidney graft failure during follow-up, and their median survival time was 5.32 years (IQR: 4.50 – 6.03 years). According to the clinical cut-off 29.1 ng / mg derived from Primary set, subjects were divided into low-risk group (uPTM-FetA / UCr< 29.1 ng / mg) and high-risk group uPTM-FetA / UCr ≥ 29.1 ng / mg) (Table 9). From Table 9, there was a significant difference in the frequency of kidney graft failure between the low-risk group and the high-risk group (4.8% and 19%, p-value< 0.001) and the survival time is significantly shorter for high-risk group than that of low-risk (5.10 [3.71, 5.80] vs 5.44 [4.93, 6.26], p-value<0.001). Table 9. Summary of 635 KTRs in 24h Set Risk group Variable Overall, Low risk, High risk, N = 6351N = 332 N = 303 p-value2Age, years, 53.00±12.77 53.09±12.71 52.91±12.86 0.863 Gender (F), n (%), 268 (42%) 135 (41%) 133 (44%) 0.457 BMI, kg / m², 26.60±4.75 26.49±4.65 26.73±4.86 0.532 eGFR, ml / min / 1.73m², 52.31±20.26 56.86±19.32 47.33±20.12 <0.001 Protein concentration, g / L, 0.10 [0.00, 0.15] 0.00 [0.00, 0.10] 0.10 [0.10, 0.30] <0.001 Protein excretion, g, 0.20 [0.00, 0.33] 0.00 [0.00, 0.23] 0.27 [0.15, 0.66] <0.001 proteinuria, n (%), 124 (20%) 28 (8.4%) 96 (32%) <0.001 Time since transplantation, years, 5.26 [1.74, 12.14] 5.56 [1.96, 12.22] 5.02 [1.56, 12.00] 0.383 Primary kidney disease, n (%), 0.508 Unknown, 101 (16%) 52 (16%) 49 (16%) Glomerulonephritis, 166 (26%) 91 (27%) 75 (25%) Interstitial-nephritis, 76 (12%) 38 (11%) 38 (13%) Cystic kidney disease, 129 (20%) 70 (21%) 59 (19%) Other congenital and hereditary kidney 33 (5.2%) 20 (6.0%) 13 (4.3%) disease, Renal vascular disease, excluding vasculitis, 35 (5.5%) 17 (5.1%) 18 (5.9%) Diabetes Mellitus, 31 (4.9%) 10 (3.0%) 21 (6.9%) Other multisystem diseases, 47 (7.4%) 26 (7.8%) 21 (6.9%) Other, 17 (2.7%) 8 (2.4%) 9 (3.0%) Acute rejection, n(%), 171 (27%) 89 (27%) 82 (27%) >0.999 HLA class I antibodies positive, n (%), 100 (16%) 48 (14%) 52 (17%) 0.409 HLA class II antibodies positive, n (%), 112 (18%) 56 (17%) 56 (18%) 0.668 Kidney donor status, n(%), 0.943Attorney Docket No.132417-0002WO01 Risk group Variable Overall, Low risk, High risk,Donor age, years, 42.86±15.41 41.40±14.94 44.49±15.78 0.013 SBP, mmHg, 135.89±17.51 135.12±17.24 136.74±17.79 0.245 DBP, mmHg, 82.41±11.09 82.21±10.81 82.63±11.41 0.635 Diabetes mellitus, n (%), 146 (23%) 69 (21%) 77 (25%) 0.197 Plasma glucose, mg / dl, 5.20 [4.80, 6.00] 5.20 [4.70, 5.80] 5.30 [4.80, 6.20] 0.010 HbA1c, %, 5.97±0.80 5.94±0.77 6.00±0.84 0.390 uPTM-FetA, ng / mL, 14.84 [7.14, 33.05] 7.49 [4.49, 11.68] 33.72 [21.00, 63.59] <0.001 uPTM-FetA / UCr, ng / mg, 27.73 [13.00, 61.01] 13.28 [8.24, 20.18] 63.08 [40.05, 105.68] <0.001 IVD103, ng / mg, 1.48±0.56 1.08±0.29 1.91±0.44 <0.001 Graft Failure, n (%), 75 (12%) 16 (4.8%) 59 (19%) <0.001 Survival Time, year, 5.32 [4.50, 6.03] 5.44 [4.93, 6.26] 5.10 [3.71, 5.80] <0.0011Mean±SD; n (%); Median [IQR]2Welch Two Sample t-test; Pearson's Chi-squared test; Kruskal-Wallis rank sum test

[0208] Figure 5 illustrates a Kaplan-Meier Survival Curve for 635 KTRs with death-censored graft failure, according to a non-limiting embodiment. Referring to Figure 5, the Kaplan-Meier estimate indicated that the KTRs in high-risk group had a significant risk of events relative to the KTRs in low- risk group, with a log-rank test p value of <0.0001. The cumulative probabilities for events in the 24h Set by baseline uPTM-FetA / UCr risk category at time points of 2-5 years after baseline were also displayed in Table 10. Table 10. Cumulative probability (with 95% confidence intervals) of event for kidney graft failure at various time points for 635 KTRs. Time points Category 2 Year 3 Year 4 Year 5 Year uPTM-FetA / UCr<29.1 1.3% 3.0% 4.2% 5.2% ng / mg (0.2% to 2.5%) (1.2% to 4.8%) (2.1% to 6.2%) (2.8% to 7.5%) uPTM-FetA / UCr≥29.1 8.9% 13% 17% 23% ng / mg (5.3% to 12%) (8.4% to 17%) (12% to 21%) (17% to 28%) Table 11. Cox Proportional Hazards Regression Models of 24h SetAttorney Docket No.132417-0002WO01(<29.1 ng / mg); The reference group for gender is male; Age and eGFR are included as continuous variables.

[0209] Further investigation with univariate Cox proportional hazards model indicated that the high- risk group had a significant risk of graft failure than the low-risk group, with a hazard ratio of 4.71 (95% CI: 2.71 – 8.19, p-value<0.001). Thus, uPTM-FetA / UCr was a significant predictor of enhance risk of kidney graft failure (Table 11). Multivariate Cox proportional hazards model adjusted for age, sex, and eGFR was also showed in Table 11. The uPTM-FetA / UCr was attenuated from the univariate analysis but remained a highly significant and independent predictor of risk for kidney graft failure with a hazard ratio of 2.80 (95% CI: 1.58 – 4.94, p-value<0.001). The reduced hazard probably reflects that uPTM-FetA / UCr may be related to the clinical factors but is clearly providing additional information.

[0210] In addition, a comparative analysis of uPTM-FetA / UCr, proteinuria and event rate using the urine samples from the 24h Set was provided to present additional interpretive information for the clinical use of uPTM-FetA / UCr (Table 12). The analysis evaluated the event rate for the primary endpoint in comparison to the uPTM-FetA / UCr (above and below the cut-off 29.1 ng / mg) and Proteinuria (total protein excretion above and below 0.5 g / d). Comparing to the reference group of uPTM-FetA / UCr <29.1 ng / mg and non-Proteinuria, hazard ratios are 3.08 (95% CI: 1.48-6.40, p- value=0.003), 6.10 (95% CI: 2.10-17.7, p-value<0.001) and 15.5 (95% CI: 7.90 – 30.5, p- value<0.001) for the second, third and fourth category, respectively.

[0211] Figure 6A illustrates a Kaplan-Meier curve for KTRs in the 24h Set with (a) non-Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment. Figure 6B illustrates a Kaplan-Meier curve for KTRs in the 24h Set with (b) Proteinuria by uPTM-FetA / UCr risk category, according to a non-limiting embodiment. Referring to Figures 6A and 6B, the Kaplan-Meier curves in Figures 6A and 6B also showed that the KTRs with uPTM-FetA / UCr ≥29.1 ng / mg had a significant risk of events relative to the low uPTM-FetA / UCr group for both non-Proteinuria and Proteinuria with log-rank test p-values 0.013 and 0.031, respectively. This suggested that uPTM-FetA / UCr can provide additional information to Proteinuria in assessing risk of kidney graft failure.Attorney Docket No.132417-0002WO01 Table 12. Hazard ratios of kidney graft failure by uPTM-FetA / UCr and Proteinuria category for 635 KTRs in the 24h Set. Category* N Number of events HR (95% CI)1p-value 1. uPTM-FetA / UCr<29.1 ng / mg & non- Reference --- Proteinuria304 112. uPTM-FetA / UCr≥29.1 ng / mg & non- Proteinuria207 21 3.08 (1.48 to 6.40) 0.0033. uPTM-FetA / UCr<29.1 ng / mg & Proteinuria28 5 6.10 (2.10 to 17.7) <0.0014. uPTM-FetA / UCr≥29.1 ng / mg & Proteinuria96 38 15.5 (7.90 to 30.5) <0.0011HR = Hazard Ratio, CI = Confidence Interval *Adjusted for age and sex

[0212] Conclusions

[0213] The results of this study demonstrate that, for KTRs, uPTM-FetA / UCr can effectively predict the risk of graft failure, independent of age, sex, and eGFR. The added prognostic value of uPTM- FetA / UCr on top of proteinuria is clear. The clinical effectiveness of the uPTM-FetA / UCr clinical cut- off (=29.1 ng / mg) was validated in an independent Validation Set and also validated in 24h Set with uPTM-FetA and urine creatinine measured by using 24-hour urine samples.

[0214] These result shows that uPTM-FetA / UCr can effectively predict the actual decrease of eGFR. Patients with high risk can take early treatment or related adjustments to achieve the effect of prevention, which has considerable clinical benefits and will be a boon for KTRs. Example 1-5

[0215] Additional marker combination models were tried and analyzed for incorporation into the kidney transplant follow up. The total of the following 4 models were tried and analyzed for predicting graft failure in KTRs: • Model1: uPTM-FetA / uCr only: • Model 2: was adjusted for age, sex, and time after transplantation at inclusion (log2), and UACR (urine albumin-to-creatinine ratio) • Model 3 was further adjusted for eGFR (estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula). • Model 4 was further adjusted for the presence of human leucocyte antigenAttorney Docket No.132417-0002WO01 Table 13. Logistic regression for graft failure in 2-years (spot urine)

[0216] Figure 7 illustrates a Receiver-operating characteristic (ROC) curve regarding uPTM- FetA / uCr and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0217] Figure 8 illustrates a Receiver-operating characteristic (ROC) curve regarding a combination of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), and UACR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0218] Figure 9 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), UACR, and eGFR and a corresponding Kaplan- Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0219] Figure 10 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), UACR, eGFR, and the presence of human leucocyte antigen class II antibodies (HLA II) and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 2 years using spot urine, according to a non-limiting embodiment.

[0220] Referring to Table 13 and Figures 7-10, combining additional marker resulted in increased AUC value for KTRs with graft failure in 2 years.Attorney Docket No.132417-0002WO01 Table 14. Logistic regression for graft failure in 5-years (spot urine)

[0221] Figure 11 illustrates a Receiver-operating characteristic (ROC) curve regarding uPTM- FetA / uCr and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0222] Figure 12 illustrates a Receiver-operating characteristic (ROC) curve regarding a combination of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), and UACR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0223] Figure 13 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), UACR, and eGFR and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0224] Figure 14 illustrates a Receiver-operating characteristic (ROC) curve of uPTM-FetA / uCr, age, sex, time after transplantation at inclusion (log2), UACR, eGFR, and the presence of human leucocyte antigen class II antibodies (HLA II) and a corresponding Kaplan-Meier curve of a marker combo model for KTRs with graft failure in 5 years using spot urine, according to a non-limiting embodiment.

[0225] Referring to Table 14 and Figures 11-14, combining additional marker resulted in increased AUC value for KTRs with graft failure in 5 years.

[0226] Intended purpose

[0227] The Human uPTM3-DKD ELISA Kit is a colorimetric immunoassay to quantify unique Fetuin-A with specific post translational modification (hereafter called as E103) in urine. This product is non-automated IVD for prognosis use and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists. The concentration of E103 should beAttorney Docket No.132417-0002WO01 corrected by urine creatinine before applying to the clinic. The Human uPTM3-DKD ELISA Kit is to be used in conjunction with clinical evaluation as an aid in assessing the prognosis of kidney function in kidney transplantation recipients.

[0228] Technical and Functional Features

[0229] The Human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrators or unknown urine samples are mixed with anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate precoated with unique PTM Fetuin-A. The monoclonal antibody recognizes unique PTM Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, the Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nm wavelength and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.

[0230] Identification Table 15. IdentificationExample 2-1

[0231] The publication, Firas F. Alkaff, et. al, "Urinary Post-Translationally Modified Fetuin-A Protein Is Associated with Increased Risk of Graft Failure in Kidney Transplant Recipients" published in Am J Nephrol 2023, available online at https: / / doi.org / 10.1159 / 000534829, is incorporated by reference herein in its entirety. The objective of the clinical performance study illustrated in Examples 2-1 through 2-5 is to evaluate the prognostic performance of the Human uPTM3-DKD ELISA test of graft failure for kidney transplantation receivers (KTR). In Examples 2-1 through 2-5, the KT data was derived from 24 hr urine.

[0232] Design and Study Population

[0233] In this prospective cohort study, we used data from the TransplantLines Food and Nutrition Biobank and cohort study (NCT02811835). All adult KTR visiting the University Medical Center Groningen (UMCG) outpatient clinic between November 2008 and March 2011 with a functioning graft for at least 1 year after transplantation, without any drug or alcohol addiction or systemic illnesses, were invited to participate in this cohort. During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participate and gave written informed con- sent. For the currentAttorney Docket No.132417-0002WO01 study, participants with missing 24h uPTM- FetA excretion measurements were excluded from the analyses. This study was conducted in accordance with the Declaration of Helsinki and Istanbul, and the study protocol was approved by the Institutional Review Board of UMCG (METc 2008 / 186).

[0234] The primary end-point of this study was graft failure, defined as the need for re- transplantation or (re-)initiation of dialysis. The secondary end-points were graft function deterioration (graft failure or doubling of serum creatinine) and all-cause mortality. For graft failure and graft function deterioration end-points, KTRs who died with a functioning graft were censored at the time of death. End-points were recorded until September 2015. With the continuous surveillance system of the UMCG outpatient clinic, no KTRs were lost to follow-up. Example 2-2

[0235] Clinical Parameters All baseline measurements were performed during a morning visit to the outpatient clinic. Blood pressure was measured twice with 15 min intervals, and then the results were averaged. Measurements were performed using a semi-automatic device (Di- namap 1846, Critikon, Tampa, USA). Bodyweight and height were measured with the participants wearing indoor clothing without shoes. Body mass index was calculated as weight in kilograms divided by height in meters squared (kg / m2), and body surface area was estimated in meters squared (m2) using DuBois and DuBois formula. Diabetes was defined according to the American Diabetes Association criteria. The estimated glomerular filtration rate (eGFR) was calculated using the creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD- EPI) formula. Primary CMV infection was defined as CMV infection occurring in a previously seronegative KTR prior to transplantation with a kidney from a seropositive donor. Secondary CMV infection was defined as CMV infection occurring in a KTR which was seropositive prior to transplantation. Relevant donor, recipient, and transplant information were extracted from the medical records, as described previously. Example 2-3

[0236] Laboratory Methods and uPTM-FetA Measurement

[0237] Blood samples were drawn at the outpatient clinic in the morning after an overnight fasting period (approximately 8–12 h), which included no medication intake. For the urine collection, all participants were instructed to collect their 24 h urine during the day before their visit.

[0238] uPTM-FetA was measured using a novel Human uPTM3-DKD ELISA kit (CE IVD Marked, Bio Preventive Medicine Corp., Hsinchu, Taiwan; Trade name: DNlite-IVD103). This assay detects post-translationally modified fetuin-A protein that contains the connecting peptide. Total urinary protein excretion was determined using the Biuret reaction (MEGA AU 150, Merck Di-agnostica, Darmstadt, Germany). Other biochemical parameters, including serum creatinine and high-sensitivity C-reactive protein (hs-CRP), were measured using routine laboratory methods.

[0239] Statistical AnalysesAttorney Docket No.132417-0002WO01

[0240] All data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R version 4.0.5 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, p value <0.05 was considered statistically significant. The distribution of continuous variables was assessed by visually inspecting the histograms and quantile-quantile plots. Normally distributed variables were presented as mean ± standard deviation, skewed variables as median (interquartile range), and categorical variables as frequency (valid percentage). Univariable linear regression analyses were performed to assess the associations between 24h uPTM-FetA excretion and clinical and biochemical parameters. After univariable analyses, we adjusted 24h uPTM-FetA excretion for sex, serum creatinine, and 24 h urinary protein excretion ≥0.5 g / 24 h. During the linear regression analyses,skewed variables were log2- transformed to fulfill the assumption for linear regression. If log2transformation could not fulfill the assumption, the variables were re- categorized into categorical variables according to the median value or acceptable cut-off levels.

[0241] Kaplan-Meier curves were used to visualize the difference in graft and patient survival among subgroups of KTR according to the median level 24h uPTM-FetA excretion (<34 μg / 24 h vs. ≥ 34 μg / 24 h), and the significances of the differences between subgroups were calculated using the log- rank test. Cox proportional hazard regression analyses were used to assess the associations of 24h uPTM- FetA excretion with graft failure, graft function deterioration, and all-cause mortality, where the associations were adjusted for potential confounders. Variables that were associated with 24h uPTM- FetA excretion in the unadjusted linear regression analyses were considered as potential confounders. In model 1, 24h uPTM-FetA excretion was adjusted for age, sex, and time after transplantation at inclusion (log2). In model 2, we additionally adjusted for eGFR. In model 3, we additionally adjusted for 24 h urinary protein excretion (log2). In model 4, we additionally adjusted KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. In model 5, we additionally adjusted for the donor characteristics (type of donor, donor age, and donor sex). In the full model (model 6), weadditionally adjusted for hs-CRP (log2).

[0242] Schoenfeld residuals on functions of time were performed to evaluate the proportional hazards assumption in the final Cox regression model of each end-point. Graft failure and graft function deterioration violated the proportional hazards assumption (p = 0.004 and p = 0.005, respectively), whereas all-cause mortality was not (p = 0.073). Therefore, Cox models with time- dependent covariates were used to calculate hazard ratios (HRs) over time for graft failure and graft function deterioration. HR was presented as per doubling of 24h uPTM-FetA excretion with 95% confidence intervals (95% CIs).

[0243] For the primary end-point, we assessed the potential interactions of age, sex, eGFR, and 24 h urinary protein excretion with 24h uPTM-FetA excretion by adding interaction terms to the full model. For the sensitivity analyses, we reevaluated the association of 24h uPTM-FetA excretion withAttorney Docket No.132417-0002WO01 graft failure after excluding the outliers. Outliers in this study were defined as values deviating morethan two standard deviations from the mean of the log224h uPTM-FetA excretion. Next to that, weused uPTM-FetA concentration and uPTM-FetA concentration indexed for creatinine (uPTM-FetA / creatinine ratio) instead of 24h uPTM-FetA excretion.

[0244] For all cross-sectional analyses, the original non-imputed dataset was used, and variables with >20 missing values (3.16%) were reported in the table footnotes. For all prospective analyses, multiple imputations using fully conditional specification were performed using the R package "mice" (number of multiple imputations = 10) to account for missing data in variables other than 24h uPTM- FetA excretion. Example 2-4

[0245] Results

[0246] Figure 15 illustrates a flowchart of the study population selection, according to a non-limiting embodiment. Referring to Figure 15, in total, 627 KTRs were included in this study. The mean age was 53 ± 13 years, 42% were female, the median time after trans- plantation was 5.3 (1.9–12.2) years,and the mean eGFR was 52 ± 20 mL / min / 1.73 m2. Median 24h uPTM-FetA excretion was 34 (17–74)μg / 24 h. Median 24 h urinary protein excretion was 0.21 (0.01–0.34) g / 24 h, of which 504 (81%) had a urinary protein excretion <0.5 g / 24 h and only 8 (1%) had a urinary protein excretion >3 g / 24 h. More detailed baseline characteristics of the study population are presented in Table 2-1. Referring to Table 2-1, Normally distributed variables were presented as mean ± standard deviation, skewed variables as median (interquartile range), and categorical variables as number (valid percentage). Smoking status was missing in 39 patients, alcohol consumption status was missing in 66 patients, CMV infection status was missing in 52 patients, Hemoglobin A1C was missing in 21 patients, and hs-CRP level was missing in 32 patients. All other variables had missing value in <20 patients. eGFR, estimated glomerular filtration rate as calculated using the creatinine-based CKD-EPI formula; HLA, human leukocyte antigen; hs-CRP, high-sensitivity C-reactive protein; mTOR, mammalian target of rapamycin; Std. β, standardized β coefficient; uPTM-FetA, urinary post-translationally modified fetuin-A; BMI, body mass index. Variables were log2transformed to fulfill the assumptions in linear regression analyses.Attorney Docket No.132417-0002WO01 Table 2-1. Baseline characteristics and linear regression analysis for 24h uPTM-FetA excretion

[0247] Figure 16 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM- FetA excretion with the serum creatine level, according to a non-lmiting embodiment. Figure 17 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the eGFR, according to a non-limiting embodiment. Figure 18 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the 24 hour albumin excretion, according to a non-limiting embodiment. Figure 19 illustrates a scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with the 24 hours urinary protein excretion, according toAttorney Docket No.132417-0002WO01 a non-limiting embodiment. Referring to Figures 16-19, 24h uPTM-FetA excretion was negatively correlated with eGFR and positively correlated with plasma creatinine concentration, urinary albumin, and 24 h urinary protein excretion.

[0248] Cross-Sectional Associations of 24h uPTM-FetA Excretion with Clinical and Biochemical Parameters

[0249] The strongest association of 24h uPTM-FetA excretion in the univariable linear regression analyses was with 24h urinary protein excretion ≥0.5 g / 24 h (standardized β coefficient [Std. β] 0.94, p < 0.001). Additionally, 24h uPTM-FetA excretion was also significantly associated with 24h urinary albumin excretion (Std. β 0.45, p < 0.001) serum creatinine (Std. β 0.34, p < 0.001), and eGFR (Std β −0.27, p < 0.001). Furthermore, female sex was also significantly associated with higher 24h uPTM- FetA excretion (Std. β −0.22, p = 0.006). Older donors, living donors, the use of proliferation inhibitors, and 24 h urinary albumin excretion were significantly associated with higher 24h uPTM- FetA excretion independent of sex, serum creatinine, and 24 h urinary protein excretion ≥0.5 g / 24 h. In contrast, longer duration after transplantation and congenital disease as the primary kidney disease were negatively associated with 24h uPTM-FetA excretion.

[0250] Prospective Analyses of the Association between 24h uPTM-FetA Excretion and Graft Failure

[0251] Table 2-2 is a prospective analysis of the association of 24 hour uPTM-FetA excretion with death-censored graft failure in 627 KTRs. Referring to Table 2-2, Cox proportional-hazard regression analyses were performed to assess the association of 24 h uPTM-FetA excretion with the risk of death-censored graft failure (i.e., the need for re-transplantation or (re-)initiation of dialysis). Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for eGFR based on creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24 h urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A. Figure 20 illustrates Kaplan-Meier curve for death-censored graft failure below and above the median of 24 h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment. Referring to Table 2-2 and Figure 20, during a median follow-up of 5.3 (4.5–6.0) years after baseline measurements, 73 (12%) KTRs developed graft failure. The most frequent cause of graft failure was chronic graft dysfunction (77%) and recurrence of primary kidney disease (10%). Other causes of graft failure include vascular problems and infection. Rates of graft failure were 4% and 19% in KTRs with baseline 24h uPTM-FetA excretion below and above the median, respectively (plog rank < 0.001). The association of 24h uPTM-FetA excretion with the riskAttorney Docket No.132417-0002WO01 for graft failure was not constant over time (pschoenfeld residue = 0.004).24h uPTM-FetA excretion was associated with graft failure within 1 year or between 1 and 3 years after baseline measurements in the unadjusted model; however, the association was lost after adjustment for confounders (HR 0.99, 95% CI: 0.77–1.27 and 0.96, 95% CI: 0.78–1.19, respectively). In contrast, 24h uPTM-FetA excretion was associated with an increased risk of graft failure after 3 years since baseline measurements (HR 1.85, 95% CI: 1.55–2.22), and the association remained significant even after adjustment for potential confounders (HR 1.31, 95% CI: 1.06–1.61) (Table 2). There was no interaction between 24h uPTM- FetA excretion and sex, age, eGFR, or 24 h urinary protein excretion for the associations of 24h uPTM-FetA excretion with graft failure (all pinteraction > 0.05). Table 2-2. Prospective analysis of the association of 24h uPTM-FetA excretion with death-censored graft failure in 627 KTRs

[0252] Sensitivity Analyses

[0253] We identified 34 (5.4%) KTRs with outliers (11 KTRs with 24h uPTM-FetA excretion <3.33 μg / 24 h and 23 KTRs with uPTM-FetA excretion >408 μg / 24 h). Table 2-3 indicates prospective analysis of the association of 24h uPTM-FetA excretion with death-censored graft failure in which outliers of 24h uPTM-FetA excretion were excluded, according to some embodiments. Referring to Table 2-3, Outliers were defined as ± 2 standard deviations from the mean of the log2-transformed 24h uPTM-FetA excretion (<3.33 µg / 24h and > 408 µg / 24h). Based on this, 34 KTR were excluded, leaving 593 KTR for Cox proportional-hazard regression analysis. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine-based CKD-EPI formula. Model 3 was further adjustedAttorney Docket No.132417-0002WO01 for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2).95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post- translationally modified fetuin-A. After the exclusion of outliers, 24h uPTM-FetA excretion remained independently associated with the risk of graft failure only after 3 years since baseline measurements (HR 1.77, 95% CI: 1.25–2.49). Table 2-3. Prospective analysis of the association of 24h uPTM-FetA excretion with death- censored graft failure in which outliers of 24h uPTM-FetA excretion were excluded.

[0254] In the other sensitivity analyses, we used uPTM-FetA concentration and uPTM- FetA / creatinine ratio instead of 24h uPTM-FetA excretion. Table 2-4 is prospective analysis of the association of uPTM-FetA concentration with graft failure in 627 kidney transplant recipients. Referring to Table 2-4, Cox proportional-hazard regression analyses were performed to assess theAttorney Docket No.132417-0002WO01 association of uPTM-FetA concentration with risk of death-censored graft failure. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2).95% CI, 95% confidence interval; HR, hazard ratio; uPTM- FetA, urinary post-translationally modified fetuin-A. Table 2-4. Prospective analysis of the association of uPTM-FetA concentration with graft failure in 627 kidney transplant recipients.

[0255] Table 2-5 indicates prospective analysis of the association of uPTM-FetA / creatinine ratio with graft failure in 627 kidney transplant recipients according to some embodiments. Referring to Table 2-5, Cox proportional-hazard regression analyses were performed to assess the association ofAttorney Docket No.132417-0002WO01 uPTM-FetA / urinary creatinine ratio with risk of death-censored graft failure. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2).95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post- translationally modified fetuin-A. Table 2-5. Prospective analysis of the association of uPTM-FetA / creatinine ratio with graft failure in 627 kidney transplant recipients.

[0256] Referring to Tables 2-3 and 2-4, similarly, uPTM-FetA concentration and uPTM- FetA / creatinine ratio were also significantly associated with graft failure only after 3 years sinceAttorney Docket No.132417-0002WO01 baseline measurements, even after adjustments for potential confounders (HR 1.35, 95% CI: 1.10– 1.65 and HR 1.36, 95% CI: 1.10–1.67, respectively)

[0257] Secondary Analyses of the Association between 24h uPTM-FetA and Graft Function Deterioration and All-Cause Mortality

[0258] During a median follow-up of 5.3 (4.0–6.0) years after baseline measurements, 121 (19%) KTRs developed graft function deterioration. Figure 21 illustrates Kaplan-Meier curve for death- censored graft function deterioration below and above the median of 24h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment. Table 2-6 indicates Supplementary Table 5. Prospective analysis of the association of 24h uPTM-FetA excretion with death-censored graft function deterioration in 627 kidney transplant recipients. Referring to Table 2-6, Cox proportional-hazard regression analyses were performed to assess the association of 24h uPTM- FetA excretion with risk of graft function deterioration. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A. Referring to Table 2-6 and Figure 21, Rates of graft function deterioration were 9% and 30% in KTRs with baseline 24h uPTM-FetA excretion below and above the median, respectively (plog rank < 0.001). The association of 24h uPTM-FetA excretion with the risk for graft failure was not constant over time (pschoenfeld residue = 0.005).24h uPTM-FetA excretion was significantly associated with an increased risk of graft function deterioration only after 3 years since baseline measurements (HR 1.64, 95% CI: 1.41–1.92), and the association remained significant even after the adjustment for potential confounders (HR 1.39, 95% CI: 1.17–1.65). Table 2-6. Prospective analysis of the association of 24h uPTM-FetA excretion with death- censored graft function deterioration in 627 kidney transplant recipients.Attorney Docket No.132417-0002WO01

[0259] During a median follow-up of 5.4 (4.9–6.1) years after baseline measurements, 132 (21%) KTRs died. Figure 22 illustrates a Kaplan-Meier curve for all-cause mortality below and above the median of 24h urinary post-translationally modified fetuin-A excretion, according to a non-limiting embodiment. There were no significant differences in the mortality rates between KTRs with 24h uPTM-FetA excretion below (19%) and above (23%) the median level (plog rank = 0.2). Table 2-7 indicates prospective analysis of the association of 24h uPTM-FetA excretion with all-cause mortality in 627 kidney transplant recipients, according to a non-limiting embodiment. Referring to Table 2-7. Cox proportional-hazard regression analyses were performed to assess the association of 24h uPTM- FetA excretion with risk of all-cause mortality. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (type of donor, donor age and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2).95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A. Unadjusted and adjusted Cox regression models showed no prospective association between 24h uPTM-FetA excretion and all-cause mortality. Table 2-7. Prospective analysis of the association of 24h uPTM-FetA excretion with all- cause mortality in 627 kidney transplant recipients.Attorney Docket No.132417-0002WO01

[0260] Discussion

[0261] In this cohort of 627 KTRs, the strongest association of 24h uPTM-FetA excretion was with 24 h urinary protein excretion ≥0.5 g / 24 h. In addition to that, shorter time after transplantation, living donor, older donor age, congenital disease as the primary kidney disease, the use of proliferation inhibitors, and 24 h urinary albumin excretion were independently associated with higher 24h uPTM-FetA excretion. In the prospective analyses, 24h uPTM-FetA excretion was independently associated with an increased risk of graft failure. However, the risk was not constant over time, with increased risk only observed beyond 3 years after baseline measurements. The association was robust in several sensitivity analyses. Similarly, 24h uPTM-FetA excretion was associated with graft function deterioration only after 3 years since baseline measurements. In contrast, 24h uPTM-FetA excretion was not associated with an increased risk of mortality.

[0262] Human fetuin-A in its single-chain precursor form comprises an A-chain, a connecting peptide, and a B-chain, in addition to a signaling peptide. Subsequently, this precursor protein undergoes post-translational modifications by means of glycosylation and proteolysis. During proteolytic processing, the connecting peptide region is removed from the precursor protein, and the single-chain form is converted into a mature two-chain form linked by a disulfide bond. Under certain conditions in which proteolysis does not occur, the single-chain fetuin-A undergoes phosphorylation instead, primarily at the serine residue in the connecting peptide region. The ELISA kit used in this study exclusively detects the latter post-translationally modified form of fetuin-A.

[0263] In healthy adults, the kidney does not express fetuin-A. However, proximal tubule epithelial cells (PTECs) develop the ability to express fetuin-A upon injury and release it to the luminal side of the tubule. Zhou et al. have shown that in cisplatin-induced and ischemia / reperfusion-induced AKI ratAttorney Docket No.132417-0002WO01 models, fetuin-A was predominantly present in the urinary exosome fraction instead of the non- exosomal fraction, indicating that fetuin-A is produced by the PTEC. Recently, Rudloff et al. also showed that PTEC can locally produce fetuin-A under hypoxic conditions after stimulation by hypoxia-inducible transcription factors. It has been postulated that the presence of fetuin-A in the proximal tubules aids in protecting the kidney from hypoxia-induced kidney inflammation by preventing the shift of macrophages to pro-inflammatory macrophages M1, and from hypoxia- induced fibrosis by antagonizing transforming growth factor-β signaling.

[0264] Both acute and chronic kidney injury are tightly associated with the occurrence of hypoxia. In the setting of kidney transplantation, the kidney is exposed to various conditions that will enhance hypoxia and hypoxic injury, such as ischemia and reperfusion during organ donation and post- operative vascular complications. Among all structures within the kidney, PTEC are the most sensitive and vulnerable as these cells are highly active and have a high oxygen demand. Because hypoxia plays a major role in the progression of kidney disease and may present in the relatively early stages of kidney injury even before the structural injury occurs, early identification of this condition may be beneficial to prevent further deterioration. In both cisplatin-induced and ischemia / reperfusion- induced AKI, urinary fetuin-A excretion increased before the surge of serum creatinine and also before the injury was present morphologically. In the current study, we found that uPTM-FetA was significantly associated with increased risk of graft failure and graft function deterioration after 3 years since baseline measurements. As one of the most important requirements for a biomarker is to reflect an underlying pathophysiology of the disease, measurement of uPTM-FetA in the urine may offer additional benefit as a biomarker for earlier detection of graft injury in KTR over currently used parameters such as serum creatinine or proteinuria.

[0265] This study has three important limitations that need to be mentioned. First, this study was performed in a single center in the Netherlands, with an over-representation of the Caucasian population. Second, as this was an observational study, residual confounding may still exist despite the number of potentially confounding factors that we have adjusted for, and the nature of this study does not allow for hard conclusions on causality. Third, we did not adjust the p values for multiple testing because this was an exploratory study. Nevertheless, this was the first study to evaluate the prospective association of urinary fetuin-A with long-term outcomes in kidney transplantation setting.

[0266] In conclusion, 24h uPTM-FetA excretion is significantly associated with lower kidney function and higher 24-h urinary albumin and protein excretion. Prospectively, 24h uPTM-FetA excretion is independently associated with an increased risk of graft failure and graft function deterioration beyond 3 years after measurements. Our findings suggest that uPTM-FetA can be used as a clinical marker to allow earlier detection of graft failure in KTR. Further studies with larger and more heterogenous KTR populations are needed to confirm our study findings. Design and Study PopulationAttorney Docket No.132417-0002WO01 Example 3-1

[0267] Design and Study Population

[0268] In this prospective cohort study, we used data from TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841). All adult KTRs visiting the University Medical Center Groningen (UMCG) outpatient clinic between November 2008 and March 2011 with a functioning graft for at least 1 year after transplantation, without any drug or alcohol addiction or systemic illnesses, were invited to participate in this cohort (18). During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participate and gave written informed consent. For the current study, participants without available 24h uC-FetA excretion measurements were excluded from the analysis. This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board (METc 2008 / 186).

[0269] The primary end-point of this study was death-censored graft function deterioration (defined as defined as doubling of serum creatinine or graft failure i.e., the need of re-transplantation or (re- )initiation of dialysis). The secondary end-point was all-cause mortality. Endpoints were recorded until September 2015. No participants were lost to follow-up. Example 3-2

[0270] Clinical parameters

[0271] All measurements were performed during a morning visit to the outpatient clinic. Blood pressure and heart rate were measured three times with 15 minutes interval, and then the results were averaged. Measurements were performed using a semi-automatic device (Dinamap 1846, Critikon, Tampa, USA). Bodyweight and height were measured with the participants wearing indoor clothing without shoes. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg / m2), and body surface area (BSA) was estimated in meters squared (m2) using DuBois and Dubois formula. Diabetes was defined according to the American Diabetes Association criteria. Estimated glomerular filtration (eGFR) was calculated using the creatinine- and cystatin C- based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Relevant donor, recipients, and transplant information was extracted from the medical records, as described previously. Example 3-3

[0272] Laboratory methods and uC-FetA measurement

[0273] Blood was drawn at the outpatient clinic in the morning after an overnight fasting period (approximately 8 to 12 hours), which included no medication intake. For the urine collection, all participants were instructed to collect their 24-hour urine during the day before their visit.

[0274] Cystatin C concentration was measured in EDTA plasma using a validated particle-enhanced turbidimetric immunoassay (Gentian, Moss, Norway). Calcification propensity test to evaluate the calciprotein particle maturation time (T50) was measured with a nanoparticle-based test. Total urinaryAttorney Docket No.132417-0002WO01 protein excretion was determined using the Biuret reaction (MEGA AU 150, Merck Diagnostica, Darmstadt, Germany). Urinary liver-type fatty acid-binding protein (L- FABP) was measured with human uL-FABP ELISA assay kit (CMIC holdings Co., Tokyo, Japan). Other biochemical parameters, including creatinine, high-sensitivity C-reactive protein (hs-CRP), and lipid profiles were measured using routine laboratory methods. The uC-FetA was measured using a novel Human uPTM3-DKD ELISA kit (Manufacturer: Bio Preventive Medicine Corp., Trade name: DNlite- IVD103).

[0275] Statistical analysesAll data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, p-value < 0.05 was considered significant. Baseline characteristics were presented as mean ± standard deviation, median [interquartile range], and frequency (percentage) for normally distributed, skewed, and nominal data, respectively. To determine whether the data were normally distributed or skewed, visual evaluation of the Q-Q plots and the histogram were performed. Univariable linear regression analyses were performed to assess the associations between uC-FetA and clinical and biochemical parameters. After univariable analyses, we adjusted uC-FetA for sex, serum creatinine, and 24h urinary protein excretion. During the linear regression analyses, skewed data were log2-transformed to fulfill the assumption for linear regression.

[0276] The association of uC-FetA with death-censored graft function deterioration and all-cause mortality were assessed with Kaplan-Meier curves, and the significance of the difference between tertiles was calculated using log-rank test. To further assess the association of uC- FetA with the outcomes, Cox-proportional hazards regression analyses were performed with adjustment for potential confounders. In model 1, 24h uC-FetA excretion was adjusted for age, sex, and time after transplantation at inclusion. In model 2, we additionally adjusted for eGFR based on creatinine- and cystatin C-based CKD-EPI formula. In model 3, we additionally adjusted for 24-hour urinary protein excretion. In model 4, we additionally adjusted for the presence of human leucocyte antigen class II antibodies, type of donor, and donor age. In model 5, we additionally adjusted for hs-CRP. In the final model (model 6), we additionally adjusted for the use of proliferation inhibitor. Schoenfeld residuals were visually checked and tested, and the final models did not violate the assumption for proportionality of hazards (p = 0.115 for death-censored graft function deterioration, and p = 0.580 for all-cause mortality). We further assessed the potential interactions of age, sex, eGFR, and 24h urinary protein excretion with 24h uC-FetA excretion by adding interaction terms to the final models. Hazard ratios are presented per doubling of 24h uC-FetA, with 95% confidence intervals (95%CI).

[0277] As we identified an interaction between uC-FetA and eGFR with death-censored graft function deterioration as the outcome, we repeated the final model of the Cox regression analyses in subgroups. We used the median eGFR value of patients with graft function deterioration (eGFR = 30 mL / min / 1.73 m2) as the cut-off to split into two groups. For all cross-sectional analyses, the originalAttorney Docket No.132417-0002WO01 non-imputed dataset was used, where variables with > 20 missing values (3.16%) are reported in the table footnotes. For all prospective analyses, multiple imputations using Fully Conditional Specification was performed using the R package 'mice' (number of multiple imputations = 10), to account for missing data among variables other than data on 24h uC-FetA excretion. Example 3-4

[0278] Results

[0279] Baseline characteristics

[0280] Figure 243 illustrates flowchart of the study participants selection according to a non-limiting embodiment. Referring to Figure 243, in total, 632 KTR were included in this study. The mean age was 53 ± 13 years, 42% were female, the median time after transplantation was 5.3 [1.7 to 12.2] years, and mean eGFR was 45.3 ±18.7 mL / min / 1.73. Median 24h uC-FetA excretion was 33.9 [17.2- 74] µg / 24h. Median 24h urinary excretion was 0.21 [0.2-0.34] g / 24h, of which 511 (81%) had urinary protein excretion < 0.5gram / 24h and 8 (1.26%) had urinary protein excretion > 3gram / 24h. More detailed baseline characteristics of the study population are presented in Table 1.Attorney Docket No.132417-0002WO01

[0281] Normally distributed data are presented as mean ± standard deviation, skewed data as median[interquartile range], and categorical data as number (valid percentage).#Variables were log2transformed to fulfill the assumptions in linear regression analyses. Diabetes was defined according to the American Diabetes Association criteria. Smoking status is missing in 39 patients, alcohol consumption status is missing in 66 patients, HbA1C is missing in 22 patients, hs-CRP level is missing in 33 patients, creatinine clearance is missing in 26 patients, urinary liver-type fatty acid binding protein is missing in 61 patients. All other variables have missing value in < 20 patients. Abbreviations: eGFR, estimated glomerular filtration rate as calculated using the creatinine and cystatin C-based CKD-EPI formula; HLA, human leucocyte antigen; hs-CRP, high-sensitivity C- reactive protein; L-FABP, liver-type fatty acid-binding protein; mTOR, mammalian target of rapamycin; uC-FetA, urinary Connecting Peptide Containing Fetuin A. Cross-sectional associations of 24h uC-FetA excretion with clinical and biochemical parameters Univariable linear regression analyses showed that male sex was significantly associated with higher 24h uC-FetA excretion (St. β = 0.11). Older donor age, living donor, and the use of proliferation inhibitor were significantly associated with higher uC-FetA excretion independent of sex, serum creatinine, and 24h urinary protein excretion. In contrast, longer duration after transplantation and higher level of circulating aspartate transaminase (AST) were negatively associated with uC-FetA excretion.

[0282] The strongest association of 24h uC-FetA excretion in the univariable linear regression analyses was with 24h urinary L-FABP excretion (St. β = 0.50) and 24h urinary protein excretion (St.Attorney Docket No.132417-0002WO01 β = 0.44). uC-FetA excretion was also positively associated with plasma NGAL and plasma Fibroblast Growth Factor 23 (FGF23) (St. β = 0.24 and St. β = 0.19, respectively), and negatively associated with eGFR and T50 (St. β = -0.29 and St. β = -0.17, respectively). After adjustment for sex, serum creatinine, and 24h urinary protein excretion, urinary L-FABP remained significantly associated with uC-FetA excretion (Std β = 0.32), whereas plasma NGAL, plasma FGF23, and T50 lost its association (Table 1). Prospective analyses of the association between 24h uC-FetA excretion and death-censored graft function deterioration. During a median follow-up of 5.3 [4.0-6.0] years after baseline measurements, 121 (19%) KTR developed graft function deterioration. Rates of graft function deterioration were 8%, 16.6%, and 32.9% in KTR with lowest, middle, and highest tertiles of baseline 24 uC-FetA excretion, respectively (plog rank < 0.001) (Figure 24). In Cox regression analyses, KTR in tertile 2 and tertile 3 of 24h uC-FetA excretion were at higher risk of graft function deterioration compared to KTR with the lowest tertile of uC-FetA excretion (HR 2.18, 95%CI: 1.22- 3.90, and HR 4.79, 95%CI: 2.82-8.14, respectively). The association of tertile 3 of 24h uC-FetA excretion remained significant after adjustment for potential confounders (HR 2.68; 95%CI, 1.49- 4.82). Similarly, 24h uC-FetA excretion was also associated with higher risk of graft function deterioration on a continuous scale, even after adjustment for potential confounders (HR per doubling 1.19, 95%CI: 1.06-1.33) (Table 2).

[0283] Table 2. Prospective analysis of the association of 24h uC-FetA excretion with death- censored graft function deterioration in 632 kidney transplant recipients.

[0284] Cox proportional-hazard regression analyses were performed to assess the association of 24h uC-FetA excretion with risk of death-censored graft function deterioration (doubling of serum creatinine or graft failure, i.e., the need of re-transplantation or (re-)initiation of dialysis). Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPIAttorney Docket No.132417-0002WO01 formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for the presence of human leucocyte antigen class II antibodies, type of donor, and donor age. Model 5 was further adjusted for serum high-sensitivity C-reactive protein (log2). Model 6 was further adjusted for the use of proliferation inhibitors. CI, confidence interval; HR, hazard ratio, uC-FetA, urinary Connecting Peptide Containing Fetuin A. Example 3-5

[0285] Sensitivity analyses

[0286] There were no interactions between 24h uC-FetA excretion and sex, age or 24h urinary protein excretion for the associations of 24h uC-FetA excretion with graft function deterioration (pinteraction = 0.07, 0.913 and 0.202, respectively). However, there was a significant interaction with eGFR (pinteraction = 0.01). Sensitivity analyses in subgroups were performed for the final model (model 6). The analyses indicated that 24h uC-FetA excretion was only significantly associated with graft function deterioration in patients with eGFR ≥ 30 mL / min / 1.73 m2 (HR 1.19, 95%CI 1.01- 1.41; p = 0.040), but not in patients with eGFR < 30 mL / min / 1.73 m2 (HR 1.02, 95%CI 0.84-1.23, p = 0.858), as presented in Figure 25. Figure 25 illustrates Forest plot for the association of uC-FetAwith death-censored graft function deterioration insubgroupsaccording to a non-limitingembodiment. Referring to Figure 25, Forest plot for the association of uC-FetA with death-censored graft function deterioration in subgroups. The model was adjusted for the full model of age, sex, log2time after transplantation at inclusion, eGFR, log224h urinary protein excretion, the presence of human leucocyte antigen class II antibodies, type of donor, donor age, log2serum high-sensitivity C- reactive protein, and the use of proliferation inhibitors. Prospective analyses of the association between uC-FetA and all-cause mortality

[0287] In secondary analyses, we assessed associations of uC-FetA with all-cause mortality. During a median follow-up of 5.4 [4.9 to 6.1] years after baseline measurements, 133 (21%) KTR died. There were no significant differences in regard to the mortality rates across the tertiles of uC- FetA (18.5% in first tertile, 20.4% in second tertile, and 24.3% in third tertile; plog rank = 0.282) (Figure 26). Unadjusted and adjusted Cox regression models also showed no prospective association between 24h uC-FetA excretion and all-cause mortality (Table 3).Attorney Docket No.132417-0002WO01

[0288] Cox proportional-hazard regression analyses were performed to assess the association of 24h uC-FetA excretion with risk of all-cause mortality. Model 1 was adjusted for age, sex, and time after transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model...

Claims

Attorney Docket No.132417-0002WO01 CLAIMS What is claimed:

1. An assay kit to determine a likelihood of a progressive renal decline, comprising: a first solution including a first reagent to interact with a post-translationally modified Fetuin- A fragments in urine fragment to indicate the level of the Fetuin A fragment in a urine sample from a recipient of a kidney transplantation after the kidney transplantation; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 20 ng / mg indicates a higher likelihood of the graft function deterioration over a period of about ten years or less, and wherein the first solution and the second solution are the same solution or different solution from each other.

2. The assay kit of claim 1, wherein the ratio being higher than about 20 ng / mg indicates a need for medical intervention to obviate the graft function deterioration.

3. The assay kit of claim 1, wherein the graft function deterioration is indicated by doubling of serum creatinine, a need of a kidney re-transplantation, or a need of dialysis.

4. The assay kit of claim 1, wherein the graft function deterioration is indicated by a decline in estimated Glomerular Filtration Rate (eGFR).

5. The assay kit of claim 1, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment.

6. The assay kit of claim 1, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine.

7. The assay kit of claim 1, wherein at least one reagent among the first reagent and the second reagent includes an antibody.

8. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg.

9. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 30 ng / mg.

10. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 35 ng / mg.

11. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 40 ng / mg.Attorney Docket No.132417-0002WO01 12. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 50 ng / mg.

13. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 70 ng / mg.

14. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg.

15. The assay kit of claim 1, wherein the higher likelihood is higher than a likelihood of graft function deterioration over the period of about ten years or less in a comparison recipient of kidney transplantation having a ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 14 ng / mg.

16. The assay kit of claim 15, wherein the second ratio being lower than about 14 ng / mg is lower than about 13 ng / mg.

17. The assay kit of claim 15, wherein the second ratio being lower than about 14 ng / mg is lower than about 9 ng / mg.

18. The assay kit of claim 15, wherein the second ratio being lower than about 14 ng / mg is lower than about 7.5 ng / mg.

19. The assay kit of claim 15, wherein the second ratio being lower than about 14 ng / mg is lower than about 7 ng / mg.

20. The assay kit of claim 15, wherein the second ratio being lower than about 14 ng / mg is lower than about 4 ng / mg.

21. The assay kit of claim 1, wherein the higher likelihood of the graft function deterioration is about 5 % or higher.

22. The assay kit of claim 1, wherein the higher likelihood of the graft function deterioration is about 10 % or higher.

23. The assay kit of claim 1, wherein the higher likelihood of the graft function deterioration is about 30 % or higher.

24. The assay kit of claim 1, wherein the graft function deterioration occurs after 2 years.

25. The assay kit of claim 1, wherein the graft function deterioration occurs after 5 years.

26. The assay kit of claim 1, wherein the device is to determine the level of the Fetuin A fragment and the level of the urinary creatinine, to assess the probability of the graft function deterioration, based on the ratio and at least one supplemental marker.

27. The assay kit of claim 26, wherein the at least one supplemental marker comprises age, sex, time after transplantation at inclusion, urine albumin-to-creatine ratio (UACR), estimated glomerular filtration rate based on creatinine- and cystatin C-based CKD-EPI formula (eGFR), a presence of human leucocyte antigen, or any combination thereof.Attorney Docket No.132417-0002WO01 28. The assay kit of claim 27, wherein the assessed probability exhibits at least about 70 percent (%) of an area under the receiver operating characteristic (ROC) curve (AUC).

29. The assay kit of claim 28, wherein the assessed probability exhibits at least about 90 percent (%) of the AUC.

30. The assay kit of claim 28, wherein the assessed probability exhibits at least about 95 percent (%) of the AUC.

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