Application of CRYL1 protein in preparation of product for evaluating postoperative recurrence risk of calcium oxalate kidney stone combined with renal papillary calcium plaque

Abstract

The application belongs to the field of biomedicine, and particularly relates to application of CRYL1 protein in preparation of products for evaluating, diagnosing or assisting in diagnosing postoperative recurrence risk of calcium oxalate kidney stone combined with renal papillary calcium plaque, and a sequence of the CRYL1 protein is shown as SEQ ID NO. 1. The application firstly finds that expression of CRYL1 protein in renal tubular and collecting duct cells of renal papillary calcium plaque tissue is reduced, and it is proved through experiments that urine CRYL1 protein is a postoperative recurrence predictor and prediction marker of patients with CaOx kidney stone combined with renal papillary calcium plaque, and a CRYL1 protein detection kit is further developed for detecting or evaluating postoperative recurrence risk of patients with CaOx kidney stone combined with renal papillary calcium plaque, which promotes personalized follow-up and prevention scheme of the patients, and has great application prospect in the clinic.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedicine, and particularly relates to application of CRYL1 protein in preparation of a product for evaluating postoperative recurrence risk of calcium oxalate kidney stone combined with renal papillary calcium plaque. BACKGROUND

[0002] Calcium oxalate kidney stone (CaOx nephrolithiasis) is a common disease of the urinary system, affecting about 8% of the population in China [1, 2] , with a high postoperative recurrence rate (about 30%-50%) in 5 years [3] . Urinary calcium, oxalate and citrate levels are affected by dietary factors, making it difficult to predict the postoperative recurrence risk of CaOx kidney stone patients [4, 5] , and other predictors reported in the literature are not ideal (AUC is less than 0.8) [6, 7] . Currently, there is no effective postoperative recurrence risk predictor for CaOx kidney stone. Randall's plaque originates from calcium salt deposition in the renal papillary interstitium, and becomes an ideal adhesion point for CaOx crystals after breaking through the renal papillary mucosa, thereby inducing CaOx kidney stones [8, 9] . Randall's plaque can be found in almost 100% of idiopathic calcium oxalate stone patients and 43% of non-calcium oxalate stone patients [10, 11] , and is considered to be the initial lesion of CaOx stone formation, which is closely related to the postoperative recurrence of CaOx kidney stone

[12] . Therefore, based on the urinary molecular markers related to Randall's plaque, it is expected to develop a postoperative recurrence risk predictor for CaOx stone patients combined with Randall's plaque.

[0003] REFERENCES

[0004] 1. Tan, S., D. Yuan, H. Su, et al., Prevalence of urolithiasis in China: a systematic review and meta-analysis . BJU Int, 2024. 133(1): 34-43.

[0005] 2. Wang, B., X. Zheng, J. Xiong, et al., Characteristics of urinary stone composition among patients with urolithiasis: a retrospective study in China .BMJ Open, 2024. 14(11): e079431.

[0006] 3. Kachkoul, R., G.B. Touimi, G. El Mouhri, et al., Urolithiasis:History, epidemiology, aetiologic factors and management . Malays J Pathol, 2023. 45(3): 333-352.

[0007] 4. Traxer, O., M. Corrales, and A. Sierra, Metabolic evaluation: isthere really a future? Curr Opin Urol, 2022. 32(4): 373-378.

[0008] 5. Coe, F.L., A. Evan, and E. Worcester, Pathophysiology-basedtreatment of idiopathic calcium kidney stones . Clin J Am Soc Nephrol, 2011. 6(8): 2083-92.

[0009] 6. Elshal, A.M., H. Shamshoun, A. Awadalla, et al., Hormonal andmolecular characterization of calcium oxalate stone formers predictingoccurrence and recurrence . Urolithiasis, 2023. 51(1): 76.

[0010] 7. Kavouras, S.A., H.G. Suh, M. Vallet, et al., Urine osmolalitypredicts calcium-oxalate crystallization risk in patients with recurrenturolithiasis .Urolithiasis, 2021. 49(5): 399-405.

[0011] 8. Evan, A.P., E.M. Worcester, F.L. Coe, et al., Mechanisms of human kidney stone formation . Urolithiasis, 2015. 43 Suppl 1: 19-32.

[0012] 9. Zhu, Z., F. Huang, M. Gao, et al., Osteogenic-Like Microenvironment of Renal Interstitium Induced by Osteomodulin Contributes to Randall's Plaque Formation . Adv Sci (Weinh), 2024. 11(40): e2405875.

[0013] 10. Randall, A., THE ORIGIN AND GROWTH OF RENAL CALCULI . Ann Surg, 1937. 105(6): 1009-1027.

[0014] 11. Matlaga, B.R., J.C. Williams, S.C. Kim, et al., Endoscopic evidence of calculus attachment to Randall's plaque . Journal of Urology, 2006. 175(5): 1720-1724.

[0015] 12. Tamborino, F., R. Cicchetti, M. Mascitti, et al., Pathophysiology and Main Molecular Mechanisms of Urinary Stone Formation and Recurrence . Int J Mol Sci, 2024. 25(5): 3075. SUMMARY

[0016] The application aims to provide application of CRYL1 protein in preparation of a product for evaluating postoperative recurrence risk of calcium oxalate kidney stones combined with renal papillary calcium plaque.

[0017] To achieve the above-mentioned object, the application adopts the following technical scheme:

[0018] The application of CRYL1 protein in preparation of a product for evaluating, diagnosing or assisting in diagnosis of postoperative recurrence risk of calcium oxalate kidney stones combined with renal papillary calcium plaque, wherein the sequence of the CRYL1 protein is shown as SEQ ID NO. 1.

[0019] CRYL1 protein (Crystallin Lambda 1, lambda-crystallin protein 1) is a kind of crystallin protein, which is an important component of the lens and plays a key role in maintaining the transparency and refractive function of the lens. In addition, CRYL1 has dehydrogenase activity, catalyzing L-gulonate to dehydro-L-gulonate, and participates in NADPH-dependent metabolic reactions (https: / / www.ncbi.nlm.nih.gov / gene / 51084). CRYL1 protein is highly expressed in the renal tubules and collecting ducts of normal kidneys, which may be related to the inhibition of renal tubular injury and renal interstitial calcium salt deposition.

[0020] The amino acid sequence of the CRYL1 protein is as follows:

[0021] massaagcvv ivgsgvigrs wamlfasggf qvklydieqq qirnalenir kemklleqagslkgslsvee qlslisgcpn iqeavegamh iqvnppyyip lvelvphpet apttvdrtha lmkkigqcpmrvqkevagfv lnrlqyaiis eawrlveegi vspsdldlvm seglgmryaf igpletmhln aegmlsycdrysegikhvlq tfgpipefsr ataekvnqdm cmkvpddpeh laarrqwrde clmrlaklks qvqpq (SEQ ID NO. 1).

[0022] The application finds through a large number of experiments that the expression of CRYL1 protein in the renal tubules and collecting duct cells of the renal papillary calcium plaque tissue is reduced, and the CRYL1 protein in the urine can be used as a predictor and a predictive marker for postoperative recurrence of calcium oxalate kidney stones combined with renal papillary calcium plaque, which has great application prospect in the clinic.

[0023] The use of substances that detect CRYL1 protein in the preparation of products for assessing, diagnosing, or assisting in the diagnosis of the risk of recurrence after surgery for calcium oxalate kidney stones with renal papillary calcification, the sequence of CRYL1 protein is shown in SEQ ID NO.1.

[0024] In one preferred embodiment, the substance for detecting CRYL1 protein includes substances that can detect CRYL1 protein content by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay.

[0025] In one preferred embodiment, the substance used to detect CRYL1 protein is a substance for binding CRYL1 protein.

[0026] In one preferred embodiment, the substance binding the CRYL1 protein is an antibody, peptide, protein, or nucleic acid molecule.

[0027] The antibody may be a monoclonal antibody, a polyclonal antibody, a genetically engineered antibody, or an antibody variable region Fv, a single-chain antibody ScFv, an antigen-binding fragment Fab or Fab', F(ab')2, Fab'-SH, or other antibody fragments, as well as antibody derivatives.

[0028] In one preferred embodiment, the antibody is a CRYL1 antibody.

[0029] Based on the same inventive concept, the present invention also claims the application of a reagent kit comprising substances for detecting CRYL1 protein, the reagent kit having at least one of the following applications:

[0030] 1) Used to detect postoperative recurrence of calcium oxalate kidney stones complicated with renal papillary calcification;

[0031] 2) Diagnosis or auxiliary diagnosis of recurrent calcium oxalate kidney stones complicated with renal papillary calcification after surgery;

[0032] 3) Assess the risk of recurrence after surgery for calcium oxalate kidney stones complicated with renal papillary calcification.

[0033] In one preferred embodiment, the substance used to detect CRYL1 protein is a CRYL1 antibody.

[0034] In one preferred embodiment, the CRYL1 antibody is a rabbit CRYL1 antibody.

[0035] In one preferred embodiment, the test sample for the kit is urine.

[0036] In one preferred embodiment, the kit further comprises CRYL1 protein as a standard.

[0037] In one preferred embodiment, the kit further comprises HRP conjugate, biotinylated detection antibody diluent, HRP conjugate diluent, wash buffer, substrate and stop solution.

[0038] The present application first discovered that CRYL1 protein is reduced in expression in renal tubular and collecting duct cells of renal papillary calcium plaque tissue, and through experiments, it has been proved that CRYL1 protein is a predictor and a predictive marker of postoperative recurrence of CaOx kidney stone combined with renal papillary calcium plaque, and a CRYL1 protein detection kit has been further developed for detecting or evaluating the risk of postoperative recurrence of CaOx kidney stone combined with renal papillary calcium plaque, which promotes personalized follow-up and prevention programs for such patients, and has great application prospects in clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a single cell sequencing result graph; wherein, Figure 1 A is an endoscopic appearance graph of normal renal papilla tissue (NRP) and renal papillary calcium plaque (RP); Figure 1 B is an operation flow chart of single cell sequencing; Figure 1 C is a UMAP dimensionality reduction clustering graph obtained by single cell sequencing of RP tissue and NRP tissue; Figure 1 D is a single cell sequencing result graph; *P<0.05; **P<0.01; ***P<0.001.

[0040] Figure 2 is a biological identification result graph of renal papillary calcium plaque (RP) tissue; wherein, Figure 2 A is a calcium salt staining (Von-Kossa) and immunohistochemical staining graph of renal papillary calcium plaque (RP) tissue; Figure 2 B is a semi-quantitative analysis result graph of immunohistochemical staining of RP and normal renal papilla tissue; Figure 2 C is an ELISA detection result graph of urine of healthy people and CaOx kidney stone combined with RP plaque patients.

[0041] Figure 3 is a standard curve graph of detection well plate 1.

[0042] Figure 4 is a standard curve graph of detection well plate 2.

[0043] Figure 5 is a standard curve graph of detection well plate 3.

[0044] Figure 6Fitting curve of urine CRYL1 protein for predicting postoperative recurrence of patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium plaque.

[0045] Figure 7 ROC curve of urine CRYL1 protein for predicting postoperative recurrence of patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium plaque.

[0046] Figure 8 ROC curve of urine CRYL1 protein for detecting 5-year postoperative recurrence rate of patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium plaque. DETAILED DESCRIPTION

[0047] The present application is not limited to the following specific embodiments, and those skilled in the art can implement the present application in other various specific embodiments according to the disclosure of the present application, or any simple changes or modifications made by using the design structure and ideas of the present application, all fall within the protection scope of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0048] In the present application, the patient inclusion criteria are as follows: (1) 18 years old ≤ age ≤ 75 years old; (2) mild hydronephrosis or no hydronephrosis; (3) no chronic kidney disease with proteinuria; (4) the renal papilla of the sampling site is more than 3 cm away from the tumor edge and is confirmed not to be invaded by the tumor by HE staining. The renal papilla tissue sample is obtained from the ex vivo specimen of the patient undergoing total nephrectomy for renal or ureteral cancer. Example 1

[0049] Single-cell sequencing

[0050] The specific steps are as follows:

[0051] (1) Tissue dissociation and single cell suspension preparation: The kidney papilla tissue sample was stored in GEXSCOPE tissue preservation solution (Singleron) and transported to Singleron laboratory (Nanjing, China, Singleron Biotech Co., Ltd.) by ice bag. The kidney papilla tissue sample was washed three times with Hanks' balanced salt solution (HBSS, Gibco, Cat# 14025-076) and then cut into 1-2 mm small pieces. Then, the tissue fragments were placed in a 15 ml centrifuge tube (Falcon, Cat# 352095), 2 ml of GEXSCOPE tissue dissociation solution (Singleron) was added, and the tissue was continuously stirred and digested at 37°C for 15 minutes. The cells were filtered through a 40 micron sterile filter screen (Falcon, Cat# 352340) and centrifuged at 300g for 5 minutes (centrifuge manufacturer: Eppendorf, Model: 5810R). After removing the supernatant, the precipitate was resuspended in 1 ml of PBS (Hyclone, Cat# SA30256.01). To remove red blood cells, 2 ml of RBC lysis buffer (Roche, Cat# 11814389001) was added to the cell suspension. The cells were centrifuged at 500xg for 5 minutes in a microcentrifuge at 15-25°C and resuspended in PBS (Hyclone, Cat# SA30256.01). After sampling, trypan blue staining (Bio-RAD, Cat# 1450013) was used and the cells were counted under a microscope (Nikon, ECLIPSE Ts2) to ensure that the concentration was 1x10^5 cells / ml and the cell viability was more than 80% before proceeding to the subsequent sample processing.

[0052] (2) Single-cell RNA sequencing details and preliminary results: The cell suspension was adjusted to a concentration of 1 x 10^5 cells per milliliter in PBS. Then, the single-cell suspension was loaded onto a microfluidic device, and scRNA-seq libraries were constructed according to the Singleron GEXSCOPE protocol using the GEXSCOPE single-cell RNA library kit (Singleron Biotechnologies). The process includes cell lysis, mRNA capture, labeling cells (barcodes) and mRNA (UMIs), reverse transcription of mRNA into cDNA and amplification, and finally fragmentation of cDNA. Each library was diluted to 4 nM and mixed for sequencing. The mixed sample was sequenced on an Illumina HiSeq X for 150 bp paired-end sequencing. Raw reads were processed by fastQC and fastp to remove low-quality reads. Poly-A tails and adapter sequences were removed by cutadapt. After quality control, the reads were further processed. The data were mapped to the reference genome GRCh38 (Ensembl version 92 annotation) using STAR software. Gene counts and unique molecular identifier (UMI) counts were obtained by featureCounts software. Based on these gene counts and UMI counts, an expression matrix file was generated for subsequent analysis.

[0053] The single-cell sequencing results are shown in Figure 1 . The results show that the expression of CRYL1 protein in renal tubular and collecting duct cells of renal papillary plaque tissue is reduced. Figure 1 A is the appearance of normal renal papilla tissue (NRP) and renal papillary plaque (RP) under endoscopy, clearly showing that the kidney stone adheres to the RP (red arrow). Figure 1 B, Figure 1 C is the UMAP dimensionality reduction clustering map obtained after obtaining the RP tissue and NRP tissue from patients undergoing total nephrectomy due to kidney tumors after approval by the Ethics Committee of Xiangya Hospital of Central South University (approval number: 201705768), performing single-cell sequencing, and displaying. Figure 1 The sequencing results of D show that CRYL1 protein is significantly underexpressed in proximal tubular epithelial cells (Proximal Tubule cells), ascending thin limb cells (Ascending Thin Limb cells), thick ascending limb cells (Thick Ascending Limb cells), unclassified tubular cells (Unassigned cells), principal cells of the collecting duct (Principal Cells), and intercalated cells (Intercalated Cells).

[0054] Von-Kossa staining

[0055] Calcium salt staining was performed on renal papillary tissue, which was divided into RP and NRP groups. The specific implementation method is as follows:

[0056] RP and NRP tissues were obtained from patients who underwent total nephrectomy due to renal tumors. The collected tissues were immediately placed in a fixative containing 10% formalin. The fixation time was usually 24 to 48 hours to ensure good tissue morphology.

[0057] (1) Section preparation:

[0058] First, paraffin embedding was performed. The steps of graphite embedding are as follows:

[0059] Dehydration: After fixation, the tissue specimens were dehydrated in gradient ethanol aqueous solutions (50%, 70%, 80%, 90%, and 100% ethanol by volume) to gradually remove water from the tissue. Each concentration of ethanol aqueous solution was usually soaked for 1 hour.

[0060] Transparency: Dehydrated tissues need to be transparentized by xylene or other transparency agents to remove alcohol.

[0061] Wax immersion: After transparency, the tissue was immersed in melted paraffin. The temperature was usually set at 60°C, and the soaking time was about 1 hour to ensure complete penetration of paraffin.

[0062] Embedding: After the tissue was completely immersed in paraffin, it was removed and placed in a mold, and cooled to room temperature to form a paraffin block.

[0063] Slicing: The steps of slicing are as follows:

[0064] Microtome settings: The paraffin block was fixed on the tray of the microtome, and the microtome was used to cut the paraffin-embedded tissue into thin sections, usually with a thickness of 4-5 microns.

[0065] Slicing process: Adjust the microtome so that the tissue surface is flat, and carefully collect the cut sections into water to prevent wrinkling or damage to the sections.

[0066] Sectioning steps: The section was taken out of the water and placed on a glass slide with adhesive force. Then the section was slowly heated to adhere to the slide.

[0067] Von-Kossa staining was performed after dewaxing and hydration of the paraffin sections. The sections were placed on a 65°C slide warmer for 2 h to allow the paraffin to melt completely and prevent the sections from falling off during subsequent staining. The dewaxing procedure was as follows: immediately after the end of the warming, the slides were placed in xylene for 15 min, and the xylene was replaced three times. The hydration procedure was as follows: after dewaxing, the slides were placed in ethanol solutions with decreasing concentrations (room temperature) for hydration. The concentrations of the ethanol solutions were as follows: absolute ethanol (to extract xylene)-absolute ethanol (to replace the container)-95% ethanol-85% ethanol-75% ethanol, 5 min each time.

[0068] (2) Staining: The slides were rinsed with double-distilled water for 3 min three times. After the distilled water around the samples was wiped off with filter paper, Von-Kossa silver solution was added dropwise using a pipette, and the slides were irradiated under a UV lamp for 30 min. Then, the staining solution was discarded, and the slides were rinsed with double-distilled water for 1 min twice. Then, the slides were stained with HE after the addition of a hypochlorite solution.

[0069] Immunohistochemical staining of CRYL1 protein was performed according to the following procedure:

[0070] (1) Section preparation: After dewaxing and hydration of the paraffin sections, the sections were subjected to immunofluorescence staining. The sections were placed on a 65°C slide warmer for 2 h to allow the paraffin to melt completely and prevent the sections from falling off during subsequent staining. Dewaxing: immediately after the end of the warming, the slides were placed in xylene for 15 min, and the xylene was replaced three times. Hydration: after dewaxing, the slides were placed in ethanol solutions with decreasing concentrations (room temperature) for hydration. The concentrations of the ethanol solutions were as follows: absolute ethanol (to extract xylene)-absolute ethanol (to replace the container)-95% ethanol-85% ethanol-75% ethanol, 5 min each time.

[0071] (2) Antigen repair: An appropriate amount of citrate antigen repair solution (PH = 6.0) was placed in a beaker, and the slides were placed in the solution after the solution was heated to boiling using a microwave oven. The slides were naturally cooled after heating for 10-15 min. The slides were rinsed with double-distilled water for 3 min twice and then immersed in PBS for 3 min three times.

[0072] (3) Blocking: After the PBS was absorbed with filter paper, 10% goat serum (animal serum of the same species as the second antibody) was added dropwise, and the slides were blocked at room temperature for 1 h.

[0073] (4) Incubation of the first antibody: After the blocking solution was absorbed, an appropriate amount of diluted (1:150 to 1:300) first antibody solution (Abcam, ab231257) was added dropwise, and the slides were incubated at 4°C overnight.

[0074] (4) Incubate the primary antibody: After removing the blocking solution, directly add an appropriate amount of primary antibody (1:200 to 1:400) incubation solution, and incubate at 4°C overnight.

[0075] (5) Incubate the secondary antibody: After removing the primary antibody incubation solution, add pre-cooled PBS and rinse for 3 times x 3 min, then add horseradish peroxidase-coupled secondary antibody dilution (1:300, Abeam) and incubate at room temperature for 1 h.

[0076] (6) DAB color development: After removing the secondary antibody incubation solution, rinse with pre-cooled PBS for 3 times x 3 min, then add DAB color development solution after removing PBS, and observe the color development under a microscope. After color development is complete, rinse with tap water for 10 min to stop color development.

[0077] (7) Hematoxylin restain: Restain with hematoxylin solution for 1-2 min, differentiate with 0.1% hydrochloric acid for 3-5 s, and then rinse with tap water for 10 min to stop staining.

[0078] (8) Mounting and observation: After dehydration, transparency, and mounting, observe under a light microscope and take pictures.

[0079] Results are shown in Figure 2 As shown in FIG. A, calcium salt staining (Von-Kossa) showed calcium salt deposition (brown-black) in the renal papillary calcium plaque (RP) tissue, and immunohistochemical staining showed that CRYL1 protein was significantly reduced in the RP tissue, indicating that CRYL1 protein was significantly reduced in the renal papillary calcium plaque tissue and urine of patients with calcium oxalate (CaOx) kidney stones.

[0080] Semi-quantitative analysis of immunohistochemical staining of RP (n=12) and normal renal papillary tissue (NRP; n=12) was performed, and the results are shown in Figure 2 B. The results showed that the staining depth was negatively correlated with the gray value.

[0081] Urine was collected from healthy individuals and patients with CaOx kidney stones combined with RP plaque, and the urine collection process was as follows:

[0082] After the patient emptied the bladder, the timing began, and the urine was not collected this time. All urine within 24 hours needed to be collected into the container. After 24 hours, the patient emptied the bladder again, and the urine needed to be collected this time. The time and urine volume needed to be recorded each time. If there was any urine missing, it needed to be noted on the record sheet. The collected urine was stored in a container, which needed to be sealed and refrigerated (2-8°C). After 24 hours, the urine was sent to the laboratory as soon as possible, and then the relevant urine analysis was performed.

[0083] ELISA was used to detect the concentration of CRYL1 protein in urine, and the CRYL1 protein / creatinine ratio (Urinary CRYL1 protein / creatinine) was calculated for correction.

[0084] The ELISA kit system is as follows:

[0085] Table 1 ELISA kit system

[0086]

[0087] In the tables, percentages are generally mass percentages, unless otherwise specified.

[0088] The process of ELISA detection is as follows:

[0089] Collect urine: The patient begins timing after emptying the bladder. No urine is collected at this time. All urine is collected in the container for the next 24 hours. After 24 hours, the patient empties the bladder again. This urine is collected. The collected urine is stored in the container, sealed and refrigerated (2-8°C). After 24 hours, the urine is sent to the laboratory as soon as possible and centrifuged at 1000 x g for 20 minutes at 2-8°C. The supernatant is collected for determination.

[0090] Note: Collect the sample in a pyrogen / endotoxin-free tube.

[0091] Samples stored at 2-8°C should be determined within 7 days, otherwise they must be divided and stored at -20°C (≤1 month) or -80°C (≤3 months). Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely and mix well before analysis (do not vortex).

[0092] If there are a large number of particulate matters in the sample, centrifuge or filter the sample before analysis.

[0093] Prepare the sample: The sample concentration should be within the range of the standard curve. As conditions may vary, the optimal dilution for each application should be determined.

[0094] All prepared samples should be used within 2 hours after dilution. It is not recommended to perform the experiment after 2 hours.

[0095] Prepare 1X wash buffer:

[0096] 1. Dilute 30 mL of wash concentrate (25X) with 720 mL of deionized or distilled water. Label as 1X wash buffer.

[0097] 2. Store the concentrate and 1X wash buffer at 2-8°C. Use the diluted buffer within 3 months.

[0098] Note: If crystals form in the concentrate, heat in a 40°C water bath and mix gently until the crystals are completely dissolved and the performance is not affected.

[0099] Prepare 1X biotinylated detection antibody solution:

[0100] Note: Working solution should be prepared immediately before use.

[0101] 1. Mix NHS-biotin with antibody at 1 : 10 to prepare biotin-coupled CRYLl antibody (100X).

[0102] 2. Calculate the amount of biotin CRYLl antibody needed (100 μL / well) before the experiment. Prepare a little more solution than the calculated amount when preparing.

[0103] 3. Centrifuge the concentrated biotinylated detection antibody at 800 x g for 1 minute at 2-8°C.

[0104] 4. Dilute the concentrated biotinylated detection antibody (100X) to IX working solution with biotinylated detection antibody diluent.

[0105] Prepare 1X HRP conjugate solution:

[0106] Note: Working solution should be prepared immediately before use.

[0107] 1. Calculate the amount needed (100 μL / well) before the experiment. Prepare a little more solution than the calculated amount when preparing.

[0108] 2. Centrifuge the concentrated HRP conjugate at 800 x g for 1 minute at 2-8°C.

[0109] 3. Dilute the concentrated HRP conjugate (100X) to IX working solution with HRP conjugate diluent.

[0110] Prepare dilution of standard:

[0111] Note: Use glass or plastic tubes to dilute the standard.

[0112] 1. Centrifuge the standard at 10,000 x g for 1 minute at 2-8°C to ensure the contents are at the bottom of the vial.

[0113] 2. Add 10 mL of sample diluent, stand for 10 minutes and invert gently several times. After complete dissolution, mix thoroughly with a pipette. This resuspension produces a 0.1 mg / mL stock solution. Add 0.4 μl of the stock solution to 1000 μl of diluent to obtain a 400 pg / ml working solution.

[0114] 3. Take 7 tubes and add 500 μL of standard and sample diluent to each tube. Pipette 500 μL of the 400 pg / mL working solution into the first tube and mix to create a 200 pg / mL working solution. Follow this procedure pipetting the solution from the previous tube into the next tube. The last tube is left blank and the solution from the previous tube is not pipetted into it. The recommended dilution gradient is as follows: 400, 200, 100, 50, 25, 12.5, 6.25, 0 pg / mL.

[0115] Allow all components to reach room temperature before use. Mix all liquid reagents before use.

[0116] Determine the number of 8-well strips needed for the assay. Insert the strips into the frame for use. Repack any unused strips and frames and store dry at -20 °C for future use. Keep the silica gel packets in the bag to keep the plates dry.

[0117] After the reagents are prepared, proceed with the assay, the assay procedure is as follows:

[0118] 1. Bind antigen:

[0119] Note: The solution should be added to the bottom of the ELISA plate well, avoiding the inner wall and creating foam as much as possible.

[0120] a. For the standard curve, add 100 μL of the standard to the appropriate well. For the sample, add 100 μL of the pre-treated sample to the well.

[0121] b. Cover the plate with plate seal and incubate at 37 °C for 90 minutes.

[0122] c. Aspirate the solution completely, do not wash.

[0123] 2. Add biotinylated detection antibody:

[0124] a. Add 100 μL of the biotinylated detection antibody working solution to each well.

[0125] b. Cover the plate with plate seal and incubate at 37 °C for 60 minutes.

[0126] c. Aspirate the solution completely and wash the wells 3 times with 350 μL of IX wash buffer. Pour off the solution from each well and add 350 μL of wash buffer to each well. Soak for 1 minute and aspirate or decant the solution in each well and blot on clean paper towels. Proceed immediately to the next step, ensuring that the wells remain moist.

[0127] 3. Add HRP conjugate:

[0128] a. Add 100 μL of the HRP conjugate working solution to each well.

[0129] b. Cover the plate with plate seal and incubate at 37°C for 30 minutes.

[0130] c. Aspirate the solution completely and repeat the washing process 5 times as described in Step 2.

[0131] 4. Add the substrate:

[0132] a. Add 90 µL of substrate reagent to each well.

[0133] b. Cover the plate with plate seal and incubate at 37°C for approximately 15 minutes. Protect the plate from light.

[0134] Note: The reaction time can be shortened or lengthened depending on the actual color change, but not more than 30 minutes.

[0135] 5. Add the stop solution:

[0136] a. Add 50 µL of stop solution to each well. This step should be performed in the same order as the substrate solution. Gently tap the side of the plate to mix.

[0137] b. The solution in the wells will change from blue to yellow.

[0138] 6. Read the plate and generate the standard curve:

[0139] 1. Pre-warm the microplate reader for approximately 15 minutes before OD measurement.

[0140] 2. Read the absorbance at 450 nm. Read the plate within 10 minutes after the addition of the stop solution.

[0141] 3. Use curve fitting software to generate the standard curve. A four-parameter algorithm provides the best standard curve fit. Under the best circumstances, the background absorbance can be subtracted from all data points (including standards, unknown samples, and controls) before plotting.

[0142] 4. Read the concentrations of unknown samples and controls from the standard curve. Multiply the values obtained for the samples by the appropriate factor to correct for sample dilution.

[0143] Note: If the OD of a sample exceeds the upper limit of the standard curve, it should be retested using appropriate dilution.

[0144] The data for the resulting standard curve is shown in the following table.

[0145] Table 2. Data for the standard curve

[0146]

[0147] The resulting standard curves for well plates 1-3 are shown in Figures Figures 3-5 , respectively.

[0148] As can be seen from Table 2, as the concentration of CRYL1 protein decreases, the OD value also gradually decreases. The OD value of the standard curve decreases from 2.4 at high concentration to about 0.07 at low concentration. According to these data, there is a negative correlation between the concentration of CRYL1 protein and the OD value, that is, the higher the concentration of CRYL1 protein, the greater the optical density (OD value). This is consistent with the standard results of ELISA test, indicating that the experimental method has good response at different concentrations. The OD values of three wells at each concentration are consistent, indicating that the experiment has good repeatability.

[0149] The urine CRYL1 protein concentration was detected by ELISA, and the CRYL1 protein / urine creatinine ratio was calculated, and the results are shown in Table 2. Figure 2 C. Statistical analysis showed that the CRYL1 protein / urine creatinine ratio of CaOx stone formers with renal papillary stones (n=25) was significantly higher than that of healthy controls (n=25), and the difference was statistically significant (P<0.001). p <0.001).

[0150] To evaluate the precision between different batches of tests, three human urine samples with low, medium and high levels of CRYL1 protein were tested 10 times in repetition.

[0151] Table 3 Repeated testing of human urine samples with low, medium and high levels of CRYL1 protein

[0152]

[0153] To evaluate the precision between different batches of tests, three human urine samples with low, medium and high levels of CRYL1 protein were tested 10 times in repetition.

[0154] Table 4 20 repeated tests of human urine samples with low, medium and high levels of CRYL1 protein

[0155]

[0156] To evaluate the stability of the test, three urine samples with high concentration of CRYL1 protein were selected and diluted (1:2; 1:4; 1:8) and then tested.

[0157] Table 5 Results of dilution test

[0158]

[0159] From Table 5, it can be seen that the floating range and average value under different dilution multiples show that the experiment for determining CRYL1 protein has good stability and consistency. The results of different dilution multiples of 1:2, 1:4 and 1:8 all show that the test method has good adaptability to CRYL1 protein samples in different concentration ranges.

[0160] A total of 316 patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium spots were tested. The inclusion criteria for patients were: (1) 18 years old ≤ age ≤ 75 years old; (2) mild hydronephrosis or no hydronephrosis; (3) no proteinuria combined with chronic kidney disease; (4) the renal papilla of the sample site is more than 3 cm away from the tumor edge and is confirmed by HE staining of the renal papilla not to be invaded by the tumor. The renal papilla tissue sample was obtained from the ex vivo specimen of the patient with renal cancer or ureteral cancer who underwent total nephrectomy. The data obtained by the test are shown in Table 5.

[0161] Table 6 Baseline characteristics of the cohort of 316 patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium spots

[0162]

[0163] Recurrence definition: CaOx kidney stone patients without residual kidney stones after lithotripsy, and X-ray, B-ultrasound or CT showed that kidney stones recurred during 5-year follow-up after surgery. The samples were collected from preoperative urine samples (frozen). The chi-square test was used for analysis of dichotomous data between groups (https: / / libguides.library.kent.edu / spss / chisquare), and the t-test was used for analysis of continuous data between groups (https: / / libguides.library.kent.edu / SPSS / IndependentTTest).

[0164] By analyzing the cohort of 316 patients with CaOx kidney stones combined with renal papillary calcium spots, it was found that traditional urine calcium, urine oxalate and urine citrate had no significant difference between the recurrence group and the non-recurrence group, while the urine CRYL1 protein / urine creatinine ratio in the recurrence group was significantly reduced (Table 6).

[0165] And the fitting curve analysis of the data is as follows:

[0166] (1) Data preparation: Ensure that the data has been preprocessed (such as removing outliers, standardizing, etc.). Determine the independent variable (X) and the dependent variable (Y).

[0167] (2) Select a model: Choose an appropriate mathematical model based on the characteristics of the data, assuming that the data follows a linear relationship.

[0168] (3) Fit the model: Use the least squares method to fit the model parameters a and b.

[0169] (4) Evaluate the model: Evaluate the fitting effect of the model by statistical indicators (such as R², RMSE, etc.). R² (coefficient of determination): Measure the proportion of data variation explained by the model. RMSE (root mean square error): Measure the deviation between predicted values and actual values.

[0170] (5) Prediction and visualization: Use the fitted model for prediction and draw the fitted curve. The results of the fitting are shown in Figure 6 . The results show that the urine CRYL1 protein concentration of patients is negatively correlated with the recurrence rate of patients 5 years after surgery, and urine CRYL1 protein is a predictor of postoperative recurrence in patients with CaOx kidney stones combined with renal papillary calcium plaque.

[0171] Urine CRYL1 protein concentration was detected in 316 patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium plaque recurrence, and urine CRYL1 protein concentration was used to predict recurrence. The ROC curve was drawn with sensitivity and specificity data, and the process was as follows:

[0172] (1) Train the model: Train a binary classification model (logistic regression) using training data.

[0173] (2) Get prediction probability: Use the model to predict the test set and get the probability that each sample belongs to the positive class.

[0174] (3) Set threshold: Select multiple thresholds from 0 to 1, and calculate TPR and FPR at each threshold.

[0175] (4) Draw ROC curve: Draw the curve with FPR as the horizontal axis and TPR as the vertical axis.

[0176] The results are shown in Figure 7 . The ROC curve shows that the area under the curve of urine CRYL1 protein concentration predicting the recurrence rate of patients 5 years after surgery is 0.87.

[0177] Randomly selected 200 patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcium plaque recurrence were detected for urine CRYL1 protein concentration, and urine CRYL1 protein content less than 86.7 pg / mg was detected as a recurrence patient. The prediction results were compared with the actual results, and the sensitivity, specificity and accuracy data were obtained. The ROC curve was drawn with sensitivity and specificity data, and the process was as follows:

[0178] (1) Train the model: Train a binary classification model (logistic regression) using training data.

[0179] (2) Get prediction probability: Use the model to predict the test set and get the probability that each sample belongs to the positive class.

[0180] (3) Set threshold: select multiple thresholds from 0 to 1, calculate TPR and FPR under each threshold.

[0181] (4) Draw ROC curve: draw the curve with FPR as the horizontal axis and TPR as the vertical axis.

[0182] The results are shown in Table 1. Figure 8 The ROC curve shows that the area under the curve of the urine CRYL1 protein concentration predicting the 5-year recurrence rate of the patients is 0.85, and the accuracy is 85.4%.

[0183] It should be noted that the above examples are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. The application of CRYL1 protein in the preparation of products for assessing, diagnosing, or assisting in the diagnosis of the risk of recurrence after surgery for calcium oxalate kidney stones complicated with renal papillary calcification, characterized in that, The sequence of the CRYL1 protein is shown in SEQ ID NO.1: massaagcvv ivgsgvigrs wamlfasggf qvklydieqq qirnalenir kemklleqagslkgslsvee qlslisgcpn iqeavegamh iqvnppyyip lvelvphpet apttvdrtha lmkkigqcpmrvqkevagfv lnrlqyaiis eawrlveegi vspsdldlvm seglgmryaf igpletmhln aegmlsycdrysegikhvlq tfgpipefsr ataekvnqdm cmkvpddpeh laarrqwrde clmrlaklks qvqpq; The 5-year recurrence rate of patients with calcium oxalate kidney stones complicated by renal papillary calcification was predicted by the ratio of urinary CRYL1 protein to urinary creatinine.

2. A reagent kit, characterized in that, The kit includes substances for detecting CRYL1 protein; the sequence of CRYL1 protein is shown in SEQ ID NO.1: massaagcvv ivgsgvigrs wamlfasggf qvklydieqq qirnalenir kemklleqagslkgslsvee qlslisgcpn iqeavegamh iqvnppyyip lvelvphpet apttvdrtha lmkkigqcpmrvqkevagfv lnrlqyaiis eawrlveegi vspsdldlvm seglgmryaf igpletmhln aegmlsycdrysegikhvlq tfgpipefsr ataekvnqdm cmkvpddpeh laarrqwrde clmrlaklks qvqpq; The 5-year recurrence rate of patients with calcium oxalate kidney stones complicated by renal papillary calcification was predicted by the ratio of urinary CRYL1 protein to urinary creatinine.

3. The reagent kit according to claim 2, characterized in that, The substance used to detect CRYL1 protein is a CRYL1 antibody.

4. The reagent kit according to claim 2, characterized in that, The substance used to detect CRYL1 protein is CRYL1 rabbit antibody.

5. The reagent kit according to claim 2, characterized in that, The test sample for the kit is urine; the kit also includes CRYL1 antibody, CRYL1 recombinant protein, HRP conjugate, biotinylated detection antibody diluent, HRP conjugate diluent, wash buffer, substrate, and stop solution.

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