A marker combination for assisting in the diagnosis of IgA nephropathy, a detection kit thereof and a non-invasive detection method for kidney injury

Abstract

The application provides a biomarker combination for non-invasive diagnosis of IgA nephropathy, comprising IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes combined with CD89. The application also provides an enzyme-linked immunoassay kit for auxiliary diagnosis of IgA nephropathy, which can jointly detect the levels of IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes combined with CD89. The application further provides an evaluation method for case damage of kidney tissue, which is based on the joint detection of the levels of IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes combined with CD89 in a sample, calculates a multi-index prediction score, and sets an evaluation threshold to determine whether short glomeruli have pathological damage. When the biomarkers are used for auxiliary diagnosis of IgA nephropathy, the biomarkers not only have the characteristics of non-invasiveness, but also have higher sensitivity, higher specificity and higher stability, and have clinical application value.

Description

Technical Field

[0001] This invention relates to biomarkers for the auxiliary diagnosis of IgA nephropathy, kits for detecting said biomarkers, and detection methods. Background Technology

[0002] IgA nephropathy (IgAN) is currently the most common primary glomerular disease worldwide. According to clinical data from China, South Korea, and Japan, IgA nephropathy accounts for approximately 30-40% of all glomerular diseases in Asian populations. In Northern Europe and the United States, it accounts for approximately 25-30%. More than 30% of these patients will progress to end-stage renal disease (ESRD) 10-20 years after onset, making IgA nephropathy one of the most common causes of uremia in young adults.

[0003] The current understanding of the pathogenesis of IgA nephropathy is generally that mucosal infection activates the mucosal immune system, which in turn activates B lymphocytes. After activation, B lymphocytes continue to differentiate and transform into plasma cells (a type of B lymphocyte) that produce and secrete IgA antibodies through T-lymphocyte-dependent and T-lymphocyte-independent pathways. During lymphocyte transport, some IgA-secreting plasma cells pathologically "hom" to other tissue areas. The IgA subtype IgA1 they secrete then escapes the antibody exocytosis process in mucosal immunity and abnormally appears in the bloodstream. Previous studies have found that circulating IgA1 molecules in patients with IgA nephropathy exhibit a deficiency of galactose-deficient IgA1 (Gd-IgA1) in its hinge region. This galactose-deficient IgA1 molecule can spontaneously aggregate or form circulating immune complexes with other proteins, such as anti-glycation antibodies against Gd-IgA1 molecules found in studies, which deposit in the glomerular mesangial area, thereby activating the complement system and inducing inflammatory responses, leading to kidney tissue damage.

[0004] The current gold standard for diagnosing IgA nephropathy remains renal biopsy. Immunopathological examination reveals the deposition of IgA-predominant immunoglobulins in the glomerular mesangial area, accompanied by varying degrees of histopathological damage. However, invasive renal biopsy carries certain risks. Many patients are unable to receive a definitive diagnosis and targeted treatment due to relative contraindications to renal biopsy (such as high bleeding risk) or because hospitals in their region or country lack the facilities for renal biopsy. Therefore, there is an urgent clinical need to develop non-invasive biomarker detection methods to aid in the diagnosis or assessment of IgA nephropathy, such as detecting abnormal IgA1 molecules in the blood, including complex polymers of specific pathological types.

[0005] The 2019 KDIGO International Consensus on Glomerulonephritis points out that although there is a great deal of research on biomarkers, there is currently a lack of biomarkers that can be effectively used in clinical practice. The following are potentially promising serum biomarkers for clinical application:

[0006] (1) Galactose-deficient IgA1 (Gd-IgA1): Gd-IgA1 is considered a potential initiating factor in the pathogenesis of IgA nephropathy. Some studies have found significantly elevated levels of galactose-deficient IgA1 in patients. For example, Moldoveanu et al., in a single-center study of 153 patients, found that Gd-IgA has good diagnostic value in Americans: with the upper limit of normal Gd-IgA1 levels as the cutoff (i.e., 90% specificity), its sensitivity was 76.5%. However, this result has not been universally confirmed in other studies. Other studies have shown that its sensitivity is only 41-57% in Japanese and Chinese populations, and the sensitivity and specificity of receiver operating characteristic (ROC) curve analysis in distinguishing between healthy controls and patients with IgA nephropathy are 41%-49% and 89%-91%, respectively. A study conducted by the Department of Nephrology at Peking University Hospital on over 1000 patients with IgA nephropathy found that the sensitivity and specificity of Gd-IgA1 in diagnosis were only 50.5% and 89.5%, respectively. Furthermore, there was significant overlap in Gd-IgA1 levels between patients and healthy individuals, highlighting its limited clinical value. In addition, this test suffers from a major technical deficiency: the currently used lectin method for measuring Gd-IgA1 is unstable, particularly exhibiting significant differences between different batches of lectin reagents, severely restricting its widespread clinical application. Currently, the method for measuring Gd-IgA1 has not been recommended by KDIGO.

[0007] (2) KM-55: In 2015, Japanese researchers prepared the monoclonal antibody KM-55 targeting glycopeptides for the determination of Gd-IgA1 levels. The results showed good consistency with the lectin HAA assay and overcame the instability of the HAA assay. However, compared to the traditional lectin HAA method, the overlap in Gd-IgA1 levels between patients and healthy controls was still significant, and this method did not outperform the traditional lectin method. The problem with this method of directly detecting Gd-IgA1 using KM-55 includes its limited detection of a single glycoform, failing to cover the diversity of IgA glycoforms. This also suggests that the clinical application prospects of Gd-IgA1 have considerable limitations.

[0008] (3) Anti-glycemic antibodies: In 2009, Suzuki et al. in the United States discovered endogenous anti-glycemic antibodies against Gd-IgA1 (where Gd-IgA1 acts as an antigen and binds to its endogenously produced anti-glycemic antibodies) and considered these anti-glycemic antibodies to be pathogenic antibodies for IgA nephropathy. They suggested that endogenous anti-glycemic antibodies could potentially serve as a specific diagnostic marker for IgA nephropathy. In a 2014 study, this group detected IgG-type anti-glycemic antibodies specific to Gd-IgA1, with a sensitivity of 89% and a specificity of 92% as a diagnostic indicator. However, this conclusion still lacks independent verification from other laboratories. Studies conducted by the inventors in the Chinese population showed that anti-glycemic antibodies were found in the serum of both IgA nephropathy patients and healthy individuals. Although the average level of anti-glycemic antibodies in patients was higher than in normal controls, if the upper limit of 90% for normal individuals was used as a threshold, less than 50% of Chinese IgA nephropathy patients had elevated levels of anti-glycemic antibodies. Therefore, the clinical value of anti-glycemic antibodies as a diagnostic marker for IgA nephropathy remains very limited.

[0009] (4) IgA / complement C3 ratio: In the early days, Japanese scholars proposed to use blood IgA or IgA / C3 ratio for the diagnosis of IgA nephropathy. Studies have shown that its sensitivity and specificity for disease diagnosis are low and its clinical value is limited.

[0010] (5) sCD89-IgA complex: Previous studies by French researchers have detected soluble CD89-IgA complexes in CD89 transgenic mice, which can deposit on mouse kidney tissue. These complexes have also been detected in the circulation of patients with IgA nephropathy and alcoholic cirrhosis. Some researchers have used soluble CD89-IgA complexes as a predictive biomarker for relapse after kidney transplantation in IgA nephropathy patients, finding that this complex level is higher in IgA nephropathy patients than in healthy controls, but the IgA-sCD89 complex level is significantly lower in relapse-free patients compared to those without relapse. Studies by Korean researchers have found that circulating CD89-IgA complexes do not predict the deterioration of kidney function in Korean IgA nephropathy patients. A recent study by Chinese researchers found elevated levels of IgA-sCD89 complexes in the circulation of IgA nephropathy patients who did not receive immunosuppressant treatment. However, there is still considerable controversy surrounding the formation of CD89-IgA complexes. Furthermore, there are currently no studies using this complex as a diagnostic biomarker for IgA nephropathy.

[0011] For the clinical diagnosis of IgA nephropathy, biomarkers used for auxiliary diagnosis should not only possess high sensitivity, high specificity, and low overlap between patients and healthy individuals, but also meet requirements such as stability and reproducibility of detection and predictive results (meaning they are unaffected by reagent changes), and ease of operation. Only in this way can they meet the needs of large-scale, non-invasive, and rapid clinical detection and diagnosis, and thus have value for industrialization and clinical application. However, as mentioned above, existing biomarkers cannot fully meet the requirements for the clinical diagnosis of IgA nephropathy. Therefore, as summarized in the latest KDIGO International Glomerulonephritis Consensus, there is still a lack of clinically applicable serological diagnostic methods for IgA nephropathy. Summary of the Invention

[0012] In view of the above background, the purpose of this invention is to provide a clinically applicable serological diagnostic method for IgA nephropathy that is not only non-invasive and has lower risk and is easier to perform than existing renal biopsy, but also has higher sensitivity, higher specificity and higher stability compared to existing biomarker-dependent diagnostic methods.

[0013] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0014] Firstly, the first aspect of the present invention provides a combination of biomarkers for non-invasive diagnosis of IgA nephropathy, including: IgA-IgG immune complex, IgG-IgA1 immune complex, and IgA immune complex that binds to CD89.

[0015] A second aspect of the present invention provides the use of a combination of immune complexes in the preparation of a product for the auxiliary diagnosis of IgA nephropathy; the combination of immune complexes includes: an IgA-IgG immune complex, an IgG-IgA1 immune complex, and an IgA immune complex bound to CD89.

[0016] The IgA-IgG immune complex described in this invention refers to IgA-IgG immune complexes that have been bound together in circulation, regardless of the binding form of IgA and IgG; this immune complex can be detected using the ELISA double antibody sandwich method, using anti-human IgA antibody as the capture antibody, and using anti-human IgG antibody as the detection antibody.

[0017] The IgG-IgA1 immune complex described in this invention refers to IgG-IgA1 immune complexes that have been bound together in circulation, regardless of the binding form of IgA1 and IgG; this immune complex can be detected using the ELISA double antibody sandwich method, using anti-human IgG antibody as the capture antibody, and using anti-human IgA1 antibody as the detection antibody.

[0018] The IgA immune complex that binds to CD89 described in this invention refers to IgA immune complexes in plasma that can bind to CD89, that is, IgA that exists in plasma in the form of a complex rather than a monomer.

[0019] Based on this, a third aspect of the present invention provides a non-invasive assessment method for pathological damage to kidney tissue, comprising:

[0020] 1) Simultaneously detect the levels of IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes bound to CD89 in samples collected from human or animal bodies to obtain measured values ​​of AG ICs, G-A1 ICs, and CD89-AICs.

[0021] 2) Based on the AG ICs, G-A1 ICs, and CD89-A ICs obtained in 1), the multi-index prediction scores are calculated using the following formula (I):

[0022] 0.127×(CD89-A ICs)+(-0.529)×(AG ICs)+0.547×(G-A1 ICs)(I)

[0023] 3) Set the evaluation threshold to 2.2456. Compare the multi-indicator prediction score calculated in 2) with the evaluation threshold. If the score is greater than or equal to the evaluation threshold, it is determined that there is a 95% prediction probability of glomerular damage; otherwise, it is determined that there is no glomerular damage.

[0024] In the evaluation method described in this invention, 1) the sample collected from a human or animal body is preferably a peripheral blood sample from a human or animal body; more preferably a peripheral blood serum sample.

[0025] In the evaluation method described in this invention, the detection method described in 1) is preferably enzyme-linked immunosorbent assay (ELISA).

[0026] A fourth aspect of the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, comprising at least an ELISA plate, a plate-covering protein, an antibody reagent, and a standard protein; wherein the antibody reagent comprises an antibody reagent required for simultaneously detecting the levels of the IgA-IgG immune complex, the IgG-IgA1 immune complex, and the IgA immune complex bound to CD89 in a sample.

[0027] In the preferred embodiment of the enzyme-linked immunosorbent assay (ELISA) kit of the present invention, the plate-covering protein includes recombinant CD89 protein, rabbit anti-human IgG polyclonal antibody, and goat anti-human IgA polyclonal antibody Fab fragment.

[0028] In a further preferred embodiment of the present invention, the recombinant CD89 protein in the enzyme-linked immunosorbent assay kit is obtained by the following method: obtaining the recombinant expression vector pcDNA-CD89-Flag using the recombinant protein sequence shown in SEQ ID No. 1 of the sequence listing; after stably transfecting HEK293 cells (ATCC) with the recombinant expression vector pcDNA-CD89-Flag, screening for cell clones that can continuously secrete CD89, and purifying the recombinant protein CD89 from the cell culture supernatant using agarose gel specifically linked with anti-flag tag antibody.

[0029] In the preferred embodiment of the enzyme-linked immunosorbent assay (ELISA) kit of the present invention, the antibody reagents include: mouse anti-human IgA-horseradish peroxidase labeled antibody, biotin-labeled mouse anti-human IgA1 heavy chain monoclonal antibody, Extra-avidin-horseradish peroxidase labeled antibody, mouse anti-human IgG heavy chain monoclonal antibody and goat anti-mouse IgG-horseradish peroxidase labeled antibody.

[0030] In the preferred embodiment of the enzyme-linked immunosorbent assay (ELISA) kit of the present invention, the standard proteins include: human IgA protein, biotin-labeled human IgA protein, and streptavidin-labeled IgG protein.

[0031] A further preferred embodiment of the enzyme-linked immunosorbent assay (ELISA) kit includes a plate-coating solution, a blocking solution, a washing solution, a chromogenic substrate, and a stop solution.

[0032] In the process of proposing this invention, we detected the levels of a large number of biomarkers that can be detected in current IgA nephropathy research, and screened out different types of IgA immune complexes detected by three different detection methods to establish a predictive model. The predictive model was established using the following method: In a population of 89 patients with IgA nephropathy and 61 healthy controls, a logistic regression model was used. The levels of five biomarkers detected during the exploration process (IgA1, Gd-IgA1, IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes bound to CD89) were used as independent variables. To use logistic regression to detect the predictive effect of independent variables on IgA nephropathy, the Box-Tidwell method was first used to statistically demonstrate a linear relationship between the continuous independent variables and the logit transformed values ​​of the dependent variable. Then, collinearity statistics were used to demonstrate that the tolerance between independent variables was greater than 0.1, the variance inflation factor was less than 10, and there was no multicollinearity among the independent variables. All independent variables were added to the model. Hosmer and Lemeshow tests showed P>0.05, indicating a high model fit. Finally, it was found that only CD89-A ICs, AG ICs, and G-A1 were significantly affected by the model. The OR risk values ​​for the three ICs variables in predicting IgA nephropathy remained statistically significant (P<0.05). Therefore, based on the given constant term, the following formula was obtained, and the calculation result was defined as the "multi-indicator prediction score".

[0033] Experiments have shown that the combined detection of the three biomarkers described in this invention has higher sensitivity and specificity for predicting the diagnosis of IgA nephropathy. We validated the model prediction in two completely independent groups, demonstrating that the model can help clinicians diagnose IgA nephropathy and guide treatment by using only peripheral blood markers. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the process for detecting the level of IgA immune complexes bound to CD89 as described in Example 2.

[0035] Figure 2 This is a schematic diagram of the process for detecting the levels of IgA-IgG immune complexes and IgG-IgA1 immune complexes in plasma samples, as described in Example 2.

[0036] Figure 3 , 4 This demonstrates the sensitivity and specificity of multi-index prediction scores in the evaluation of Example 2, as well as the determination of cutoff values ​​using prediction scores.

[0037] Figure 5 This reflects the area under the ROC curve for different biomarker evaluation methods.

[0038] Figure 6 This reflects the overlap between patients (IgAN) and the normal population (HC) who used different biomarker assessment methods.

[0039] Figure 7 This reflects the area under the ROC curve for each of the six evaluation methods in the comparative example.

[0040] Figure 8 This reflects the area under the ROC curve for each evaluation method in Comparative Example 7.

[0041] Figure 9 This reflects the area under the ROC curve for each of the eight evaluation methods in the comparison example. Detailed Implementation

[0042] To facilitate understanding, the present invention will be further illustrated below by listing embodiments, but the present invention is not limited to the listed embodiments.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0044] Example 1. Enzyme-linked immunosorbent assay kit for clinical auxiliary diagnosis of IgA nephropathy

[0045] The enzyme-linked immunosorbent assay kit for the clinical auxiliary diagnosis of IgA nephropathy specifically includes:

[0046] 1. Plate coating solution: pH 9.6 bicarbonate buffer

[0047] 2. Platelet-forming protein:

[0048] Recombinant human CD89 protein;

[0049] Rabbit anti-human IgG polyclonal antibody;

[0050] Fab fragment of goat anti-human IgA polyclonal antibody;

[0051] 3. Antibody detection:

[0052] Mouse anti-human IgA-horseradish peroxidase labeled antibody (abcam, ab7383);

[0053] Biotin-labeled mouse anti-human IgA1 heavy chain monoclonal antibody (southern);

[0054] Extra-avidin-horseradish peroxidase-labeled antibody (Sigma);

[0055] Mouse anti-human IgG heavy chain monoclonal antibody (Abcam);

[0056] Goat anti-mouse IgG-horseradish peroxidase labeled antibody;

[0057] 4. Standard protein:

[0058] Commercial human IgA protein (Abcam, ab91020);

[0059] Commercially available biotin-labeled human IgA protein;

[0060] Commercially available streptavidin-labeled IgG protein;

[0061] 5. Blocking buffer, samples, standards, and diluent for detection antibodies: 1% BSA / 0.05% PBST solution;

[0062] 6. Washing solution: 0.05% PBST solution;

[0063] 7. Colorimetric solutions: Peroxidase substrate colorimetric solutions A and B;

[0064] 8. Termination solution: 1M concentrated sulfuric acid;

[0065] 9. Enzyme-linked immunosorbent assay (ELISA) plate: 96-well plate with high protein binding.

[0066] Example 2. Non-invasive assessment method for pathological damage to renal tissue

[0067] The evaluation samples were plasma from patients with IgA nephropathy and healthy individuals, sourced from the Department of Nephrology at Peking University Hospital. Patients were those diagnosed with IgA nephropathy via renal biopsy who had signed informed consent, while healthy individuals were healthy blood donors who had signed informed consent.

[0068] Using the enzyme-linked immunosorbent assay kit described in Example 1, the levels of IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes bound to CD89 in plasma samples collected from humans were simultaneously detected. The specific detection methods are as follows:

[0069] I. Detection of IgA immune complex levels binding to CD89, the general procedure is as follows: Figure 1 As shown, the specific steps include:

[0070] 1. Plate coating: Take an ELISA plate, dilute recombinant human CD89 protein to a concentration of 2.5 μg / ml with plate coating solution, add 50 μl to each well, coat overnight at 4℃ to obtain CD89 coated plate, wash 3 times with washing buffer for 1 minute each time.

[0071] 2. Blocking: Add 1% BSA / PBST blocking solution, 100 μl / well, to the CD89 coated plate prepared in step 1, block at 37°C for 1 h, and wash 3 times with washing solution for 1 minute each time.

[0072] 3. Sample addition: 50 μl of plasma sample to be tested (the plasma sample is diluted 1:1000 with 1% BSA / PBST buffer), incubate at 37°C for 3 h, and wash 3 times with washing buffer for 1 minute each time.

[0073] 4. Add antibody: After completing step 3, add 50 μl of HRP-labeled anti-IgA antibody to each well, dilute it 1:1000 with 1% BSA / PBST buffer, and incubate at 37°C for 1 h.

[0074] 5. TMB color development: Prepare the peroxidase substrate TMB color development solution fresh at a 1:1 ratio, and protect it from light. When washing the plate, mix the AB solution in the shaker in the dark. Add 50 μl of color development solution to each well and develop the color at room temperature in the dark for 30 min.

[0075] 6. Termination of Reading: Reading was terminated with the stop solution. The microplate reader was used for dual-wavelength readings at 450 / 570nm. Commercially available human IgA protein was serially diluted as a standard. The corresponding concentrations were calculated using a Logistic four-parameter fitted standard curve. The equation for the Logistic four-parameter fitted standard curve is as follows:

[0076] y = (AD) / [1 + (x / C)^B] + D; where A = 2.07884, B = -1.08659, C = 1854.70355, D = 0.00030. The standard curve equation is r. 2 =0.99994.

[0077] The OD value obtained by the above steps is substituted into the y value in the standard curve equation to calculate the x value, which is used to evaluate the level of IgA complex that can bind to CD89 in the patient's plasma sample, and the predictive index CD89-AICs is obtained.

[0078] II. Detection of IgA-IgG immune complex and IgG-IgA1 immune complex levels in plasma samples:

[0079] 1. Plate coating: Take an ELISA plate, dilute the rabbit anti-human polyclonal IgG antibody / goat anti-human IgA polyclonal antibody Fab fragment to a concentration of 2.5 μg / ml with the plate coating solution, add 50 μl to each well, and coat overnight at 4℃ to obtain IgG antibody and IgA antibody coated plates. Wash 3 times with washing buffer for 1 minute each time.

[0080] 2. Blocking: Add 1% BSA / PBST blocking solution (100 μl / well) to the IgG and IgA antibody coated plates prepared in step 1, block at 37°C for 1 h, and wash 3 times with washing solution for 1 minute each time.

[0081] 3. Sample addition: 50 μl of plasma sample to be tested (the plasma sample is diluted 1:100,000 with 1% BSA / PBST buffer), incubate at 37°C for 1 h, and wash 3 times with washing buffer for 1 minute each time.

[0082] 4. Add detection antibodies (primary antibodies): After completing step 3, for IgG antibody-coated plates, add 50 μl of Biotin-labeled mouse anti-human IgA1 antibody to each well, diluted 1:4000 with 1% BSA / PBST buffer; for IgA antibody-coated plates, add 50 μl of mouse anti-human IgG heavy chain monoclonal antibody to each well, diluted 1:2000 with 1% BSA / PBST buffer. Incubate all coated plates at 37°C for 1 h.

[0083] 5. Add secondary antibody: After completing step 4, for IgG antibody-coated plates, add 50 μl of Extra-avidin-horseradish peroxidase-labeled antibody to each well, and dilute 1:2000 with 1% BSA / PBST buffer; for IgA antibody-coated plates, add 50 μl of goat anti-mouse IgG-horseradish peroxidase-labeled antibody to each well, and dilute 1:4000 with 1% BSA / PBST buffer. Incubate all coated plates at 37°C for 1 h.

[0084] 6. TMB color development: Prepare the peroxidase substrate TMB color development solution fresh at a 1:1 ratio, and protect it from light. When washing the plate, mix the AB solution in the shaker in the dark. Add 50 μl of color development solution to each well and develop the color at room temperature in the dark for 30 min.

[0085] 7. Termination of Reading: Stop reading using a stop solution. Use a dual-wavelength microplate reader at 450 / 570nm. Standard proteins are prepared by serial dilution of commercially available biotin-labeled IgA protein and streptavidin-labeled IgG protein. Calculate the corresponding concentrations using a Logistic four-parameter fitted standard curve. The equation for the Logistic four-parameter fitted standard curve is as follows:

[0086] IgG antibody-coated plate: y = (AD) / [1 + (x / C)^B] + D; where A = 1.48699, B = -0.97439, C = 18.25720, D = -0.00767. Standard curve equation r 2 =0.99967.

[0087] IgA antibody-coated plate: y = (AD) / [1 + (x / C)^B] + D; where A = 1.61149, B = -1.21433, C = 93.22440, D = 0.00374. Standard curve equation r 2 =0.99200.

[0088] Substitute the OD degree obtained from the above steps into the y value in the standard curve equation to calculate the x value, and obtain the prediction indices AG ICs and G-A1 ICs.

[0089] III. Calculating Multi-Indicator Prediction Scores

[0090] Based on the AG ICs, G-A1 ICs, and CD89-A ICs obtained in steps one and two, the multi-index prediction score A is calculated using the following formula (I):

[0091] A=0.127×(CD89-A ICs)+(-0.529)×(AG ICs)+0.547×(G-A1 ICs) (I)

[0092] IV. Differentiation of Pathological Injuries to Kidney Tissue

[0093] Studies have found that, for example Figure 3 , 4 As shown, when the predicted score is greater than 2.2456, the method of the present invention has a specificity of 95.1% for judging glomerular pathological damage, while the sensitivity is still 67.4%. Therefore, an evaluation threshold of 2.2456 is set, and the multi-index predicted score A calculated in step three is compared with the evaluation threshold. If the score is greater than or equal to the evaluation threshold, it is judged as glomerular pathological damage; otherwise, it is judged as non-glomerular pathological damage.

[0094] Comparative Example 1.

[0095] Using the same plasma sample as in Example 2 as the evaluation sample, the level of IgA-IgG immune complexes was detected separately (the detection method is the same as step two in Example 2). The area under the ROC curve is shown in [Figure 1]. Figure 5 .like Figure 5 As shown by the single-dot dashed “AG_ICs”, its specificity and sensitivity were 88.52% and 32.58%, respectively; the overlap between patients and the normal population was 75.3% (e.g., Figure 6 (AG ICs).

[0096] Comparative Example 2.

[0097] Using the same plasma sample as in Example 2 as the evaluation sample, the level of IgG-IgA1 immune complexes was detected separately (the detection method is the same as step two in Example 2). The area under the ROC curve is shown in [Figure 1]. Figure 5 .like Figure 5 As shown by the straight line “GA_ICs”, its specificity and sensitivity were 80.33% and 85.39%, respectively; the overlap between patients and the normal population was 27.0% (e.g., Figure 6 (G-AICs in the text).

[0098] Comparative Example 3.

[0099] Using the same plasma sample as in Example 2 as the evaluation sample, the level of IgA immune complexes binding to CD89 was detected separately (the detection method is the same as step one in Example 2). The area under the ROC curve is shown in [Figure 1]. Figure 5 .like Figure 5 As shown in the midline "CD89_binding_IgA_ICs", its specificity and sensitivity were 83.61% and 69.66%, respectively; the overlap between patients and the normal population was 39.3% (e.g., Figure 6 (CD89-A ICs).

[0100] Comparative Example 4.

[0101] Using the same plasma sample as in Example 2 as the evaluation sample, the level of IgA1 was detected separately (the detection method is described in the reference: Moldovanu Z, Wyatt RJ, JY Lee, et al. Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels[J]. Kidney International, 2007, 11(11):1148-1154). The area under the ROC curve is shown in [reference missing]. Figure 5 .like Figure 5 As shown by the dashed line "IgA1", its specificity and sensitivity were 90.16% and 60.67%, respectively; the overlap between patients and the normal population was 40.4% (e.g., ...). Figure 6 (PlasmaIgA1 in the text).

[0102] Comparative Example 5.

[0103] Using the same plasma sample as in Example 2 as the evaluation sample, the level of Gd-IgA1 was detected separately (KM55 detection kit). The area under the ROC curve is shown in [Figure 1]. Figure 5 .like Figure 5As shown by the double-dotted line “Gd_IgA1”, its specificity and sensitivity were 75.41% and 75.53%, respectively; the overlap between patients and the normal population was 60.7% (e.g., Figure 6 (Gd-IgA1 in the text).

[0104] Comparative Example 6.

[0105] Using the same plasma sample as in Example 2 as the evaluation sample, the levels of IgA immune complexes binding to CD89 and IgG-IgA1 complexes were detected (detection method as described in step one of Example 2). The levels were compared with traditional indicators such as IgA1 level, Gd-IgA1 level, and IgG-IgA1 complex level alone. The area under the ROC curve was as follows: Figure 7 As shown, the area under the ROC curve was 0.883, and the specificity and sensitivity were 93.44% and 80.9%, respectively.

[0106] Comparative Example 7.

[0107] Using the same plasma sample as in Example 2 as the evaluation sample, the levels of IgA immune complexes binding to CD89 and IgG-IgA1 complexes were detected (using the same detection method as described in step one of Example 2). The levels of IgA-IgG complexes were also detected in combination. The area under the ROC curve is shown in Figure 1. Figure 8 As shown, the area under the ROC curve increased to 0.911 compared to Comparative Example 6.

[0108] Comparative Example 8.

[0109] Using the same plasma sample as in Example 2 as the evaluation sample, in addition to detecting the three IgA complexes (the detection method is the same as step one in Example 2), the levels of Gd-IgA1 and IgA1 were detected separately. After combination, it was found that the area under the ROC curve was as follows: Figure 9 As shown, it did not significantly improve diagnostic efficacy.

[0110] In summary, the combined detection of the three biomarkers of this invention for the assessment of pathological damage to renal tissue and the diagnosis of IgA nephropathy has better sensitivity and specificity than other existing biomarkers. At the same time, the overlap between patients and healthy individuals is low, the detection method is simple, and the detection results are stable and reproducible. sequence list <110> Peking University First Hospital <120> A combination of biomarkers for the auxiliary diagnosis of IgA nephropathy, its detection kit, and a non-invasive method for detecting kidney injury. <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 249 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Pro Leu Gly Thr Thr Leu Leu Cys Leu Val Leu Cys Leu Gly 1 5 10 15 Gly Ala Ile Gly Ala Gly Gly Gly Ala Pro Pro Met Pro Pro Ile Ser 20 25 30 Ala Leu Ser Ser Pro Val Ile Pro Leu Ala Gly Ser Val Leu Ile Gly 35 40 45 Cys Gly Ala Ile Ala Gly Ala Thr Leu Thr Gly Leu Met Ile Ile Leu 50 55 60 Ala Ser Thr Thr Ala Gly Ile Gly Ala Ala Leu Leu Pro Thr Ala Gly 65 70 75 80 Thr Ala Pro Gly Pro Val Ile Ala His Met Ala Ala Ala Leu Ala Gly 85 90 95 Ala Thr Gly Cys Gly Thr Ala Ile Gly His Thr Ala Pro Ala Thr Ser 100 105 110 Ala Thr Leu Gly Leu Val Val Thr Gly Leu Thr Gly Leu Pro Pro Leu 115 120 125 Ser Ala Ala Ala Gly Leu Val Leu Met Pro Gly Gly Ala Ile Ser Leu 130 135 140 Thr Cys Ser Ser Ala His Ile Pro Pro Ala Ala Pro Ser Leu Ala Leu 145 150 155 160 Gly Gly Gly Leu Ser Leu Pro Gly His Gly Ser Gly Gly His Pro Ala 165 170 175 Ala Pro Ser Leu Gly Pro Val Ala Leu Ala Val Ser Gly Ile Thr Ala 180 185 190 Cys Thr Gly Thr Thr Ala Ala Ser Pro Thr Leu Thr Ser Pro Pro Ser 195 200 205 Ala Ala Leu Gly Leu Val Val Thr Ala Ser Ile His Gly Ala Thr Thr 210 215 220 Thr Gly Ala Ala Thr Leu Ala His Ala Gly Ala Thr Leu Ala His Ala 225 230 235 240 Ile Ala Thr Leo Ala Ala Ala Ala Leo 245

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, comprising at least an ELISA plate, a plate-covering protein, an antibody reagent, and a standard protein; wherein the antibody reagent comprises an antibody reagent required for simultaneously detecting IgA-IgG immune complexes, IgG-IgA1 immune complexes, and IgA immune complexes binding to CD89 in a sample; wherein the IgA immune complexes binding to CD89 refer to IgA immune complexes in plasma that can bind to CD89, i.e., IgA existing in plasma in complex form rather than monomeric form.

2. The kit according to claim 1, characterized in that: The IgA-IgG immune complex refers to the circulating IgA-IgG immune complex that can be detected using the ELISA double-antibody sandwich method, with anti-human IgA antibody as the capture antibody and anti-human IgG antibody as the detection antibody; the IgG-IgA1 immune complex refers to the circulating IgG-IgA1 immune complex that can be detected using the ELISA double-antibody sandwich method, with anti-human IgG antibody as the capture antibody and anti-human IgA1 antibody as the detection antibody; the IgA immune complex that binds to CD89 refers to IgA immune complexes in plasma that can bind to CD89, that is, IgA existing in plasma in complex form rather than monomeric form.

3. The kit according to any one of claims 1-2, characterized in that: The antibody reagents include: mouse anti-human IgA-horseradish peroxidase labeled antibody, biotin-labeled mouse anti-human IgA1 heavy chain monoclonal antibody, Extra-avidin-horseradish peroxidase labeled antibody, mouse anti-human IgG heavy chain monoclonal antibody and goat anti-mouse IgG-horseradish peroxidase labeled antibody.

4. The kit according to any one of claims 1 or 2, characterized in that: The plate-forming proteins include recombinant CD89 protein, rabbit anti-human IgG polyclonal antibody, and goat anti-human IgA polyclonal antibody Fab fragment; the standard proteins include: human IgA protein, biotin-labeled human IgA protein, and streptavidin-labeled IgG protein.

5. The kit according to any one of claims 1-2, characterized in that: It also includes plate coating solution, blocking solution, washing solution, chromogenic substrate and stop solution.

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