Method, device and equipment for detecting HLA-DSA negative antibody-mediated rejection in kidney transplantation

Abstract

The present invention belongs to the field of biomedical technology, and discloses a method and device for detecting HLA-DSA-negative antibody-mediated rejection reaction in kidney transplantation, a device, an equipment, and a storage medium. When the HLA-DSA of the kidney transplantation subject is negative, the non-HLA antibody level of the subject to be tested is further judged, that is, the relative level value of each marker (including one or more of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody) in the serum of the HLA-DSA-negative subject to be tested is obtained, and the detection value of the subject to be tested is calculated according to the relative level value of each marker and a preset coefficient. If the detection value is greater than a specified cutoff value, it is determined that the subject to be tested suffers from antibody-mediated rejection reaction, thereby further distinguishing between T cell-mediated rejection reaction and antibody-mediated rejection reaction when the HLA-DSA of the kidney transplantation subject to be tested is negative, and the detection specificity and sensitivity can be improved on the basis of non-invasive and simple detection of HLA-DSA-negative antibody-mediated rejection reaction in kidney transplantation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a method and device, equipment, storage medium for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation, and application of a non-HLA antibody marker. Background Art

[0002] Allogeneic renal transplantation is currently the first choice for the treatment of end-stage renal disease. Rejection after renal transplantation is the main reason affecting the function of transplanted kidney and eventually leading to the failure of transplanted kidney. Transplant rejection includes T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR, AMR). The two complement each other and jointly affect the function of transplanted kidney. Acute ABMR is the main cause of early transplanted kidney function loss in renal transplantation, and it is easy to develop into chronic antibody-mediated rejection, resulting in poor long-term renal transplantation results.

[0003] With the development of clinical testing technology, preoperative human leukocyte antigen (HLA) matching reduces the occurrence of HLA-mediated cellular rejection and HLA antibody-mediated humoral rejection; preoperative panel reactive antibodies (PRA) detection and complement-dependent lymphocytotoxicity (CDC) test can diagnose the patient's sensitization state early and reduce the occurrence of hyperacute and acute accelerated rejection; the development of immunosuppressive drugs and the optimization of immunosuppressive regimens have improved the treatment effect of cell-mediated rejection, all of which have improved the short-term survival rate of transplanted kidneys. However, nearly half of the transplanted kidneys still fail 10-15 years after surgery, that is, the long-term survival rate of transplanted kidneys has not been significantly improved.

[0004] The long-term survival rate of transplanted kidneys is mainly affected by ABMR. Compared with TCMR, ABMR can cause patients to have more severe clinical symptoms, and is difficult to treat, has a poor prognosis, and is prone to recurrence. It has become a key issue that needs to be tackled in the field of organ transplantation. Transplanted kidney biopsy is the gold standard for diagnosing antibody-mediated rejection of kidney transplants, but the results of biopsy are largely dependent on the subjective experience of the examiner, which is prone to missed diagnosis and misdiagnosis. In addition, biopsy is invasive and expensive, which many kidney transplant recipients cannot accept.

[0005] Clinically, kidney transplant recipients are usually treated according to ABMR when their serum creatinine level is elevated, their urine protein is positive or their urine volume is decreased, color Doppler ultrasound shows decreased transplanted kidney blood flow, and their donor-specific anti-HLA antibodies (HLA-DSA) are positive. However, about half of kidney transplant recipients are HLA-DSA negative when they develop ABMR.

[0006] Clinically, kidney transplant recipients with elevated serum creatinine levels, positive urine protein or decreased urine volume, reduced transplanted kidney blood flow as indicated by color Doppler ultrasound, and negative HLA-DSA are often treated according to TCMR, and the treatment of TCMR is mainly hormone shock, or combined with rabbit anti-human thymocyte immunoglobulin (ATG) or anti-human T cell immunoglobulin (ALG). The treatment of ABMR according to the treatment of TCMR is often not ideal, causing irreversible damage to the transplanted kidney, or even loss of transplanted kidney function.

[0007] Therefore, non-invasive and simple detection of ABMR is of great significance for prolonging the survival time of transplanted kidneys. Although there is a system for non-invasive detection of rejection reactions based on the levels of cytokines or chemokines and their receptors in the serum of kidney transplant recipients, it cannot distinguish between TCMR and ABMR, and its specificity and sensitivity are low, making it difficult to guide clinical symptomatic treatment. Summary of the invention

[0008] The purpose of the present invention is to provide a method and device, equipment, storage medium for detecting HLA-DSA negative antibody-mediated rejection reaction in renal transplantation, as well as an application of a non-HLA antibody marker, which can not only non-invasively and simply detect HLA-DSA negative antibody-mediated rejection reaction in renal transplantation, but also improve the detection specificity and sensitivity.

[0009] The first aspect of the present invention discloses a method for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation, comprising:

[0010] Obtaining the relative level values ​​of each marker in the serum of the HLA-DSA negative test subject;

[0011] According to the relative level value of each marker and the preset coefficient, the test value of the object to be tested is calculated by the following formula:

[0012]

[0013] Among them, P AMR represents the detection value, n is the number of markers, n is any positive integer from 1 to 6, w i represents the preset coefficient of the ith marker, E irepresents the relative level value of the i-th marker, and n markers include one or more of the following six non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody;

[0014] If the detected value is greater than a specified cutoff value, a prompt message is issued to indicate that the subject to be tested suffers from an antibody-mediated rejection reaction.

[0015] In some embodiments, if the detected value is less than or equal to the specified cutoff value, a prompt message is issued to indicate that the subject to be tested has not had an antibody-mediated rejection reaction.

[0016] In some embodiments, the preset coefficients of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody are 0.299, 0.404, 1.222, 0.343, 0.452, and 0.775, respectively.

[0017] In some embodiments, the specified cutoff value is 2.842.

[0018] In some embodiments, obtaining the relative level values ​​of each marker in the serum of an HLA-DSA negative test subject comprises:

[0019] Obtaining the median fluorescence intensity of each marker in the serum of the HLA-DSA negative test subject;

[0020] The half value of the ratio between the median fluorescence intensity of each marker and the corresponding average value of the median fluorescence intensity of healthy subjects was calculated as the relative level value of each marker.

[0021] The second aspect of the present invention discloses the use of a non-HLA antibody marker in preparing a kit for detecting HLA-DSA negative antibody mediated rejection reaction in renal transplantation or constructing a detection model for HLA-DSA negative antibody mediated rejection reaction in renal transplantation, wherein the non-HLA antibody marker comprises at least one combination of the following six non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody.

[0022] The third aspect of the present invention discloses a device for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation, comprising:

[0023] An acquisition unit, used to acquire the relative level value of each marker in the serum of the HLA-DSA negative test subject;

[0024] The calculation unit is used to calculate the detection value of the object to be tested according to the relative level value of each marker and the preset coefficient through the following formula:

[0025]

[0026] Among them, P AMR represents the detection value, n is the number of markers, n is any positive integer from 1 to 6, w i represents the preset coefficient of the ith marker, E i represents the relative level value of the i-th marker, and n markers include one or more of the following six non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody;

[0027] The prompt unit is used to issue a prompt message indicating that the subject to be tested suffers from an antibody-mediated rejection reaction when the detection value is greater than a specified cutoff value.

[0028] The fourth aspect of the present invention discloses an electronic device, comprising a memory storing executable program code and a processor coupled to the memory; the processor calls the executable program code stored in the memory to execute the method for detecting HLA-DSA negative antibody mediated rejection reaction in kidney transplantation disclosed in the first aspect.

[0029] The fifth aspect of the present invention discloses a computer-readable storage medium storing a computer program, wherein the computer program enables a computer to execute the method for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation disclosed in the first aspect.

[0030] The beneficial effects of the present invention are that the provided method and apparatus, equipment, storage medium for detecting HLA-DSA-negative antibody-mediated rejection reaction in renal transplantation, and application of a non-HLA antibody marker, further determine the non-HLA antibody level of the subject to be tested when the HLA-DSA of the subject to be tested is negative, that is, obtain the relative level value of each marker (including one or more of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody) in the serum of the HLA-DSA-negative subject to be tested, and calculate the detection value of the subject to be tested according to the relative level value of each marker and a preset coefficient. If the detection value is greater than the specified cutoff value, it is determined that the subject to be tested suffers from antibody-mediated rejection reaction, thereby further distinguishing between T cell-mediated rejection reaction and antibody-mediated rejection reaction when the HLA-DSA of the subject to be tested is negative, and the detection specificity and sensitivity can be improved on the basis of non-invasive and simple detection of HLA-DSA-negative antibody-mediated rejection reaction in renal transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings herein show specific examples of the technical solutions described in the present invention, and together with the specific implementation methods, constitute a part of the specification, and are used to explain the technical solutions, principles and effects of the present invention.

[0032] Unless otherwise specified or defined, the same reference numerals in different drawings represent the same or similar technical features, and the same or similar technical features may also be represented by different reference numerals.

[0033] Figure 1 is the relative levels of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody in the stable recipients and ABMR recipients in the examples of the present invention;

[0034] Figure 2 is a receiver operating characteristic curve diagram of the detection model established in the embodiment of the present invention;

[0035] Figure 3 The P AMR The change curve of

[0036] Figure 4 It is a flow chart of a method for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation in an embodiment of the present invention;

[0037] Figure 5 It is a schematic structural diagram of a device for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation according to an embodiment of the present invention;

[0038] Figure 6 It is a structural schematic diagram of an electronic device in an embodiment of the present invention.

[0039] Description of reference numerals:

[0040] 501, acquisition unit; 502, calculation unit; 503, prompt unit; 601, memory; 602, processor. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings.

[0042] Unless otherwise specified or defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In combination with the technical solution of the present invention in a realistic scenario, all technical and scientific terms used herein may also have meanings corresponding to the purpose of implementing the technical solution of the present invention. The "first, second..." used herein is only used to distinguish the names and does not represent a specific quantity or order. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0043] Unless otherwise specified or defined, the “said” and “the” used in this document refer to the technical features or technical contents mentioned or described before the corresponding position, and the technical features or technical contents may be the same as or similar to the technical features or technical contents mentioned therein.

[0044] Undoubtedly, technical contents or technical features that are contrary to the purpose of the present invention or are obviously contradictory should be excluded.

[0045] Clinically, non-HLA antibodies are also found to be involved in antibody-mediated rejection of kidney transplantation. Compared with stable recipients, the levels of some non-HLA antibodies in recipients with HLA-DSA negative antibody-mediated rejection are significantly increased, such as angiotensin Ⅱ type 1 receptor (Angiotensin Ⅱ Receptor 1, AT1R) antibodies, glutathione S-transferase θ1 (GSTT1) antibodies, etc. HLA-DSA negative antibody-mediated rejection is basically related to non-HLA antibodies. Therefore, the establishment of a detection model for HLA-DSA negative antibody-mediated rejection of kidney transplantation based on non-HLA antibody levels has important clinical application value.

[0046] In order to facilitate the understanding of the present invention, firstly, the establishment and verification process of a renal transplantation HLA-DSA negative antibody mediated rejection reaction detection model provided by the present invention is described.

[0047] The main equipment used include: ELx800 absorbance microplate reader (BioTek), Luminex 200 detection system (Luminex), diaphragm pump (Millipore), 5810R centrifuge (Eppendorf), MIX-3000 mixing elf (Hangzhou Miou Instrument Co., Ltd.), and MIX-25 mini mixer (Hangzhou Miou Instrument Co., Ltd.).

[0048] The main reagents used include: Anti-Angiotensin Ⅱ Receptor 1-Antibodies ELISA Kit (CellTrend), LIFECODES Non-HLA Antibody Kit (Immucor), and LIFECODESHLA-LSA Kit (Immucor).

[0049] The main consumables used include: vacuum blood collection tubes (Jiangsu Kangjian Medical Supplies Co., Ltd.) and FiltrEX 96-well filter plates (Corning).

[0050] Data analysis software used: IBM SPSS Statistics 22 statistical software.

[0051] 1. The process of establishing a model for detecting HLA-DSA negative antibody-mediated rejection in renal transplantation

[0052] (1) Collection of serum samples and basic information of recipients: Serum samples from 22 renal transplant recipients with HLA-DSA negative antibody-mediated rejection at the time of biopsy diagnosis and serum samples from 44 stable renal transplant recipients after surgery were collected and stored at -80°C. The basic information of these recipients is shown in Table 1.

[0053] Table 1 Basic information of recipients with HLA-DSA negative antibody-mediated rejection and stable recipients of renal transplantation

[0054]

[0055]

[0056]

[0057] The quantitative data are expressed as mean ± standard deviation. DBD: brain-dead donor. DCD: cardiac-dead donor. DBCD: brain-cardiac-dead donor.

[0058] (2) Detection of AT1R antibody levels in the above serum by ELISA: Take out the required components from the kit 30 minutes in advance, thaw at room temperature, shake for 15 seconds before use, and centrifuge instantly; dilute the 10× cleaning solution 10 times with ddH2O, and dilute the serum sample 100 times with sample diluent before the experiment; add 100 μL of the standard and diluted sample to the test wells of a 96-well plate coated with AT1R antigen, and incubate at 4°C for 2 h; pat the test wells dry on absorbent paper, add 300 μL of 1× cleaning solution, incubate for 1 min, and pat dry, and wash 3 times in total; add 100 μL of 1× cleaning solution to each test well, and incubate for 1 min. Add 100 μL of secondary antibody coupled to horseradish peroxidase (HRP) to the test wells and incubate at room temperature at 300 rpm for 1 hour; pat the test wells dry on absorbent paper, add 300 μL of 1× cleaning solution, incubate for 1 minute and then pat dry, and wash 3 times in total; add 100 μL of tetramethylbenzidine (TMB) colorimetric solution to each test well, incubate at room temperature at 300 rpm for 20 minutes in the dark, and then add 100 μL of reaction stop solution to each test well; use an enzyme-labeled instrument to detect the absorbance of the test wells to 450 nm light, make a standard curve, and calculate the AT1R antibody level in the serum samples.

[0059] (3) Detection of 60 non-HLA antibody levels using the single antigen microbead method: Take out the required components from the kit in advance and equilibrate to room temperature; Tear off the tin foil covering the wells to be used in the 96-well filter plate, add 200 μL ddH2O to each well, leave at room temperature for 5 min, and slowly remove the liquid with a vacuum pump; Shake and mix the non-HLA microbeads, add 40 μL of microbeads to each well, then add 10 μL of serum sample, negative control serum, and positive control serum to each well, and pat to mix; Wrap the 96-well filter plate with tin foil and incubate at room temperature at 300 rpm for 30 min; Add 100 μL of Add 250 μL of cleaning solution to each well, tap to mix, and slowly remove the liquid with a vacuum pump; add 250 μL of cleaning solution to each well, tap to mix, and slowly remove the liquid with a vacuum pump, and then wash twice with 250 μL of cleaning solution in sequence; dilute the phycoerythrin-coupled goat anti-human antibody stock solution 10 times with the cleaning solution, add 50 μL of phycoerythrin-coupled goat anti-human antibody dilution solution to each well, wrap the 96-well filter plate with tin foil, and incubate at room temperature at 300 rpm for 30 minutes; prepare the Luminex 200 detection system, start it up and maintain it, and according to the reaction pattern diagram, select the corresponding reading template to edit the on-machine detection pattern; after the incubation, add 150 μL of cleaning solution to each well, mix it evenly with a pipette, and immediately detect it on the machine.

[0060] Among them, 60 non-HLA antibodies include: ACTIN antibody, AGRN antibody, APOL2 antibody, ARHGDIB antibody, ATP5B antibody, CCP antibody, CD40 antibody, CGB5 antibody, collagen I antibody, collagen II antibody, collagen III antibody, collagen IV antibody, collagen V antibody. Antibodies to CXC chemokine ligand 11 (CXCL11) and CXC chemokine ligand 9 (CXCL9), anti-dexamethasone-induced protein (DEXI), anti-endomucin (EMCN), anti-α-enolase (ENO1), anti-Fas cell surface death receptor (FAS), and anti-fibroblast growth factor 2 (GFAP). 1) Antibodies, polyleucine fibronectin-like transmembrane protein 2 (FLRT2) antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, glial cell line-derived neurotrophic factor (GDNF) antibody, glutathione S-transferase θ1 (GSTT1) antibody, histidyl tRNA synthetase (HARS) antibody, heat shock protein B1 (HSPB1) antibody, intercellular adhesion molecule 1 (ICAM1) antibody, interferon gamma (IFNG) antibody, interleukin 21 (IL21) antibody, interleukin 8 (IL8) antibody, keratin 18 (KRT18) antibody, keratin 8 (KRT8) antibody, galectin 3 (LGALS3) antibody, galectin 8 (LGALS8) antibody, lamin A / C (LMNA) antibody, spider toxin receptor 1 (LPHN1) antibody, myosin (Myosin) antibody, nucleolin (NCL) antibody, purine receptor P2Y11 (P2RY11) antibody, peroxisome trans-2-enoyl-CoA reductase (PECR) antibody, phospholipase A2 receptor 1 (PLA2R1) antibody, protein kinase C η (PRKCH) antibody, protein kinase Cζ (PRKCZ) antibody, O-type protein tyrosine phosphatase receptor (PTPRO) antibody, receptor tyrosine kinase-like orphan receptor 1 (ROR1 antibody), SHC-transforming protein 3 (SHC3) antibody, small nuclear ribonucleoprotein polypeptide B2 (SNRPB2) antibody, small nuclear ribonucleoprotein polypeptide N (SNRPN) antibody, Sjögren's syndrome antigen B (SSB) antibody, signal transducer and activator of transcription 6 (STAT6) antibody, thyroglobulin (Thyroglobulin) antibody, transferrin (Transferrin) antibody, microtubule protein alpha 1b (TUBA1B) antibody, microtubule protein beta class I (TUBB) antibody, microtubule protein (TUBULIN) antibody, focal adhesion protein (VCL) antibody, vascular endothelial growth factor A (VEGFA) antibody and vimentin (VIM) antibody.

[0061] (4) Biomarker screening: The relative levels of 60 non-HLA antibodies were calculated based on the MFI averages of non-HLA antibodies in healthy subjects provided by the LIFECODES Non-HLA Antibody Kit. The single-sample KS test was used to verify whether the ATI1R antibody levels and the relative levels of 60 non-HLA antibodies in stable recipients and recipients with antibody-mediated rejection were in accordance with normal distribution. The data that were in accordance with normal distribution were statistically analyzed using the t-test to determine whether there were significant differences between the two groups of recipients. The data that were not in accordance with normal distribution were statistically analyzed using the Mann-Whitney U test. Statistical analysis found that the relative levels of Collagen I antibody, Collagen II antibody, CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, SNRPB2 antibody, SSB antibody, Thyroglobulin antibody, TUBB antibody, and VEGFA antibody were significantly different between the two groups of recipients (P<0.001).

[0062] (5) Establishment of detection model: Logistic stepwise regression analysis was used to establish an optimization model based on the relative levels of 12 non-HLA antibodies that were significantly different between the two groups of recipients (P < 0.001). The optimal detection model for HLA-DSA negative antibody-mediated rejection in renal transplantation was P AMR = 0.299 × E CSF +0.404×E FLRT2 +1.222×E GAPDH +0.343×E GSTT1 +0.452×E PRKCZ +0.775×E SSB , where P AMR Represents the efficacy of this non-HLA antibody combination in detecting HLA-DSA negative antibody-mediated rejection in renal transplantation, ECSF 、E FLRT2 、E GAPDH 、E GSTT1 、E PRKCZ 、E SSB They represent the relative levels of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody in serum. The relative level of an antibody refers to half of the ratio of the median fluorescent intensity (MFI) measured by the antibody to the average MFI of the antibody in healthy people.

[0063] The renal transplantation HLA-DSA negative antibody mediated rejection detection model established in this example was analyzed by the receiver operating characteristic curve. The results are as follows: Figure 2 As shown. Figure 2 It can be concluded that the area under the receiver operating characteristic curve is 0.913 (P < 0.001). AMR When the cutoff value is 2.842, the sensitivity is 0.875 and the specificity is 0.841. It can be understood that the above-screened optimal 6 non-HLA antibodies can also be used to prepare a kit for detecting HLA-DSA negative antibody-mediated rejection in renal transplantation.

[0064] 2. Validation process of the HLA-DSA negative antibody-mediated rejection detection model for renal transplantation

[0065] (1) Collection of serum samples: Use red-tipped blood collection tubes to collect peripheral blood from kidney transplant recipients with abnormally elevated serum creatinine or positive urine protein. Let the blood stand at room temperature for 30 min to allow the blood to coagulate naturally. Centrifuge at 3000 × g for 5 min and dispense the serum into 1.5 mL centrifuge tubes.

[0066] (2) Detection of HLA antibodies by single antigen microbead method: Take the LSA microbead mixture out of the refrigerator and thaw it at room temperature away from light, then place it on ice away from light; equilibrate the cleaning solution at room temperature, and place other reagents at 2-8°C away from light; tear off the tin foil covering the wells to be used in the 96-well filter plate, add 200 μL ddH2O to each well, leave it at room temperature for 5 min, and slowly remove the liquid with a vacuum pump; shake the LSA microbead mixture for 1 min to mix it thoroughly, add 40 μL of microbeads to each well, and then add 10 μL of serum sample, negative control serum and positive control serum to each well, respectively, and pat to mix; wrap the 96-well filter plate with tin foil and incubate it at room temperature at 300 rpm for 30 min; add 100 μL of Washing solution, tap to mix, and slowly remove the liquid with a vacuum pump; add 250μL of washing solution to each well, tap to mix, and slowly remove the liquid with a vacuum pump, and wash twice with 250μL of washing solution in sequence; dilute the phycoerythrin-coupled goat anti-human antibody stock solution 10 times with washing solution, add 50μL of phycoerythrin-coupled goat anti-human antibody dilution solution to each well, wrap the 96-well filter plate with tin foil, and incubate at room temperature at 300rpm for 30min; prepare the Luminex 200 detection system, start it and maintain it, and select the corresponding reading template to edit the on-machine detection pattern according to the reaction pattern diagram; after the incubation, add 150μL of washing solution to each well, mix it with a pipette, and immediately test it on the machine; determine whether HLA-DSA is positive based on the HLA antibody test results and the donor's HLA type; if HLA-DSA is positive, the clinician determines whether antibody-mediated rejection occurs based on experience. If HLA-DSA is negative, non-HLA antibody levels need to be tested.

[0067] (3) Detection of non-HLA antibody levels by single antigen microbead method: Take out the required components from the kit in advance and equilibrate to room temperature; tear off the tin foil covering the wells to be used in the 96-well filter plate, add 200 μL ddH2O to each well, leave at room temperature for 5 min, and slowly remove the liquid with a vacuum pump; oscillate to mix the non-HLA microbeads, add 40 μL of microbeads to each well, then add 10 μL of serum sample, negative control serum, and positive control serum to each well, and pat to mix; wrap the 96-well filter plate with tin foil and incubate at room temperature at 300 rpm for 30 min; add 100 μL of Add 250 μL of cleaning solution to each well, tap to mix, and slowly remove the liquid with a vacuum pump; add 250 μL of cleaning solution to each well, tap to mix, and slowly remove the liquid with a vacuum pump, and then wash twice with 250 μL of cleaning solution in sequence; dilute the phycoerythrin-coupled goat anti-human antibody stock solution 10 times with the cleaning solution, add 50 μL of phycoerythrin-coupled goat anti-human antibody dilution solution to each well, wrap the 96-well filter plate with tin foil, and incubate at room temperature at 300 rpm for 30 minutes; prepare the Luminex 200 detection system, start it up and maintain it, and according to the reaction pattern diagram, select the corresponding reading template to edit the on-machine detection pattern; after the incubation, add 150 μL of cleaning solution to each well, mix it evenly with a pipette, and immediately detect it on the machine.

[0068] (4) Detection of HLA-DSA negative antibody-mediated rejection in renal transplantation: Based on the average MFI of non-HLA antibodies in healthy people provided by the LIFECODES Non-HLAAntibody Kit, the relative levels of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody were calculated and introduced into the renal transplantation HLA-DSA negative antibody-mediated rejection detection model to calculate the detection value P AMR When P AMR When P > 2.842, it indicates that the kidney transplant recipient suffers from antibody-mediated rejection. AMR When ≤2.842, it indicates that the kidney transplant recipient has no antibody-mediated rejection reaction.

[0069] (5) Treatment and treatment effect of kidney transplant recipients with HLA-DSA negative antibody-mediated rejection detected by this detection model: According to the kidney transplant HLA-DSA negative antibody-mediated rejection detection model, 36 kidney transplant recipients were detected with antibody-mediated rejection; these recipients underwent plasma exchange or immunoadsorption, followed by infusion of immunoglobulin, and the transplanted renal function of 19 recipients returned to normal, while the transplanted renal function of 17 recipients did not improve significantly; the non-HLA antibodies of recipients whose transplanted renal function did not improve significantly were detected, and it was found that the relative levels of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody did not decrease significantly. These recipients were treated with rituximab or bortezomib, and the transplanted renal function returned to normal; the non-HLA antibody levels of these 36 recipients were also tested when they improved after treatment. Compared with the time of diagnosis, the P AMR Significantly reduced, such as Figure 3 shown.

[0070] (6) Biopsy diagnosis of kidney transplant recipients who were not tested for antibody-mediated rejection: Based on the renal transplant HLA-DSA negative antibody-mediated rejection detection model, the diagnosis of antibody-mediated rejection in 15 kidney transplant recipients was not established; biopsy was performed on these 15 kidney transplant recipients, and it was found that 5 of them were diagnosed with focal segmental glomerulosclerosis (FSGS), 3 were diagnosed with T cell-mediated rejection, 2 were diagnosed with membranous nephropathy, 2 were diagnosed with mesangial proliferative glomerulitis, 1 was diagnosed with thin basement membrane nephropathy, 1 was diagnosed with IgA nephropathy, and 1 was diagnosed with ischemic renal injury.

[0071] In summary, the present invention collects serum from kidney transplant recipients, detects AT1R antibody levels and other 60 non-HLA antibody levels, screens out non-HLA antibodies with significant differences (P<0.001) between recipients with HLA-DSA negative antibody-mediated rejection and stable recipients through statistical analysis, and uses Logisti regression analysis to obtain a detection model using one or more of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody as markers. The application method is simple in material collection, can detect HLA-DSA negative antibody-mediated rejection of kidney transplantation in a non-invasive, efficient, economical and accurate manner, and has good clinical application value.

[0072] In clinical practice, it is only necessary to bring the relative level values ​​of one or more of the CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody in the serum of HLA-DSA negative recipients with abnormal transplant renal function into the detection model, and the calculation results can be used to determine whether the recipient has an antibody-mediated rejection reaction. Figure 4 As shown, the embodiment of the present invention discloses a method for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation that can be applied to clinical detection, comprising the following steps S10-S50:

[0073] S10. Obtain the relative level values ​​of each marker in the serum of the HLA-DSA negative test subject.

[0074] First, peripheral blood can be collected from subjects with abnormal transplant renal function (excessively high serum creatinine levels, positive urine protein, or low urine volume) to separate the serum. Then, the single antigen microbead method is used to detect HLA antibodies in the serum, and combined with the HLA typing results of the subject to be tested, it is determined whether the serum contains HLA-DSA. If the HLA-DSA in the serum is negative, the single antigen microbead method is used to detect the levels of various markers in the serum to test the subject for antibody-mediated rejection.

[0075] Specifically, step S10 includes the following steps S101-S102:

[0076] S101. Obtain the median fluorescence intensity of each marker in the serum of an HLA-DSA negative test subject.

[0077] S102. Calculate half the value of the ratio between the median fluorescence intensity of each marker and the corresponding average value of the median fluorescence intensity of healthy subjects as the relative level value of each marker.

[0078] S20, according to the relative level value of each marker and the preset coefficient, the detection value of the object to be tested is obtained by weighted calculation using the following formula (1):

[0079] (1)

[0080] Among them, P AMR represents the detection value, n is the number of markers, n is any positive integer from 1 to 6, w i represents the preset coefficient of the ith marker, E i represents the relative level value of the i-th marker, and the n markers include one or more of the following six non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody.

[0081] It is understandable that the above 6 non-HLA antibodies can be used alone as markers for detecting HLA-DSA negative antibody-mediated rejection of renal transplantation. In different embodiments, one or more combinations thereof can be used for detection, without limitation. In this embodiment, 6 antibodies are preferably used for detection, i.e., n=6.

[0082] Since the preset coefficients of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody are 0.299, 0.404, 1.222, 0.343, 0.452, and 0.775, respectively, the optimal renal transplantation HLA-DSA negative antibody-mediated rejection detection model can be obtained by substituting all six non-HLA antibodies as markers into formula (1). AMR =0.299×E CSF +0.404×E FLRT2 +1.222×E GAPDH +0.343×E GSTT1 +0.452× E PRKCZ +0.775×E SSB .

[0083] Among them, E CSF 、E FLRT2 、E GAPDH 、E GSTT1 、E PRKCZ 、E SSB They respectively represent the relative levels of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody and SSB antibody in serum.

[0084] S30, determine whether the detection value is greater than the specified cutoff value. If yes, execute step S40; otherwise, execute step S50.

[0085] Preferably, the specified cutoff value can be set to 2.842. AMR When P > 2.842, it indicates that the renal transplant candidate suffers from antibody-mediated rejection. AMR When ≤2.842, it indicates that the kidney transplant candidate has not experienced antibody-mediated rejection reaction.

[0086] S40: If the detected value is greater than the specified cutoff value, a prompt message is issued to indicate that the subject to be tested suffers from an antibody-mediated rejection reaction.

[0087] S50: If the detected value is less than or equal to the specified cutoff value, a prompt message is issued to indicate that the object to be tested has not undergone antibody-mediated rejection reaction.

[0088] In summary, compared with the prior art in which T cell-mediated rejection is directly used for treatment when the renal transplant is HLA-DSA negative, which results in unsatisfactory results, the embodiments of the present invention can further determine the non-HLA antibody level of the subject to be tested when the HLA-DSA of the renal transplant is negative, and perform antibody-mediated rejection detection on the subject to be tested in combination with the non-HLA antibody level, thereby further distinguishing T cell-mediated rejection from antibody-mediated rejection. Therefore, not only can HLA-DSA-negative antibody-mediated rejection of renal transplantation be detected non-invasively and simply, but also the detection specificity and sensitivity can be improved. Moreover, the operation is simple and the cycle is short, which improves the detection efficiency, and is conducive to the early diagnosis and symptomatic treatment of HLA-DSA-negative antibody-mediated rejection, and prolongs the survival time of the transplanted kidney. In addition, the number of markers is small, the cost required is low, and it is easier to be accepted by patients.

[0089] like Figure 5 As shown, the embodiment of the present invention discloses a device for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation, comprising an acquisition unit 501, a calculation unit 502 and a prompt unit 503, wherein:

[0090] An acquisition unit 501 is used to acquire the relative level value of each marker in the serum of an HLA-DSA negative test subject;

[0091] The calculation unit 502 is used to obtain the detection value of the object to be detected by weighted calculation according to the relative level value of each marker and the preset coefficient through the following formula:

[0092]

[0093] Among them, P AMR represents the detection value, n is the number of markers, n is any positive integer from 1 to 6, w i represents the preset coefficient of the ith marker, E i represents the relative level value of the i-th marker, and n markers include one or more of the following six non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody;

[0094] The prompt unit 503 is used to issue a prompt message indicating that the subject to be tested suffers from an antibody-mediated rejection reaction when the detection value is greater than a specified cutoff value.

[0095] The prompt unit 503 is further used to issue a prompt message indicating that the object to be tested has not had an antibody-mediated rejection reaction when the detection value is less than or equal to a specified cutoff value.

[0096] The acquisition unit 501 may include the following subunits (not shown):

[0097] An acquisition subunit, used to acquire the median fluorescence intensity of each marker in the serum of an HLA-DSA negative test subject;

[0098] The calculation subunit is used to calculate half the value of the ratio between the median fluorescence intensity of each marker and the corresponding average value of the median fluorescence intensity of healthy persons as the relative level value of each marker.

[0099] like Figure 6 As shown, an embodiment of the present invention discloses an electronic device, including a memory 601 storing executable program codes and a processor 602 coupled to the memory 601;

[0100] The processor 602 calls the executable program code stored in the memory 601 to execute the method for detecting the HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation described in the above embodiments.

[0101] The embodiments of the present invention further disclose a computer-readable storage medium storing a computer program, wherein the computer program enables a computer to execute the method for detecting HLA-DSA negative antibody-mediated rejection reaction in kidney transplantation described in the above embodiments.

[0102] The purpose of the above embodiments is to exemplarily reproduce and deduce the technical solution of the present invention, and to fully describe the technical solution, purpose and effect of the present invention. Its purpose is to make the public understand the disclosed content of the present invention more thoroughly and comprehensively, and it does not limit the scope of protection of the present invention.

[0103] The above embodiments are not exhaustive enumerations of the present invention, and there may be multiple other implementations not listed. Any replacement and improvement made without violating the concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of non-HLA antibody markers in constructing an antibody-mediated rejection detection model for HLA-DSA negative renal transplant recipients, characterized in that: The non-HLA antibody markers include the following 6 non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody.

2. Use of a non-HLA antibody marker in the preparation of a kit for detecting antibody-mediated rejection in HLA-DSA negative renal transplant recipients, characterized in that: The non-HLA antibody markers include the following 6 non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody.

3. A device for detecting HLA-DSA negative antibody-mediated rejection in kidney transplantation, characterized in that: include: An acquisition unit, used to acquire the relative level value of each marker in the serum of the HLA-DSA negative test subject, wherein the relative level value refers to half of the ratio of the median fluorescence intensity measured by the marker to the average median fluorescence intensity of the marker in healthy people; The calculation unit is used to calculate the detection value of the object to be tested according to the relative level value of each marker and the preset coefficient through the following formula: Among them, P AMR represents the detection value, n is the number of markers, n is 6, w i represents the preset coefficient of the ith marker, E i represents the relative level value of the i-th marker, and the n markers include the following 6 non-HLA antibodies: CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody; among them, the preset coefficients of CSF2 antibody, FLRT2 antibody, GAPDH antibody, GSTT1 antibody, PRKCZ antibody, and SSB antibody are 0.299, 0.404, 1.222, 0.343, 0.452, and 0.775, respectively; The prompt unit is used to issue a prompt message for indicating that the subject to be tested suffers from an antibody-mediated rejection reaction when the detection value is greater than a specified cutoff value, and the specified cutoff value is set to 2.

842.

4. The device for detecting HLA-DSA negative antibody-mediated rejection in kidney transplantation according to claim 3, characterized in that: The acquisition unit comprises: An acquisition subunit, used to obtain the median fluorescence intensity of each marker in the serum of an HLA-DSA negative test subject; The calculation subunit is used to calculate half the value of the ratio of the median fluorescence intensity of each marker to the average value of the median fluorescence intensity of the marker in healthy people as the relative level value of each marker.

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