Application of ASGPR1 binding type IgA in IgA nephropathy diagnosis and treatment effect prediction marker
The ELISA detection method for ASGPR1-binding IgA has solved the problems of stability and efficacy in the diagnosis and treatment of IgA nephropathy, achieving efficient diagnosis and treatment outcome prediction while reducing the risks of invasive examinations.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
- Filing Date
- 2026-02-09
- Publication Date
- 2026-05-12
AI Technical Summary
Current technologies lack stable and efficient biomarkers for the diagnosis and prediction of IgA nephropathy. Invasive renal biopsy procedures are frequent and high-risk. Proteinuria level diagnosis lacks specificity, and existing indicators have poor sensitivity and specificity.
ASGPR1-binding IgA (ASGPR1-IgA) was used as a biomarker. The level of ASGPR1-binding IgA in plasma was detected by enzyme-linked immunosorbent assay (ELISA) to establish a stable detection method. The high affinity between recombinant ASGPR1 protein and polyIgA was used for diagnosis and prediction of treatment efficacy.
It improves the diagnostic efficacy of IgA nephropathy. The area under the ROC curve of ASGPR1-binding IgA is significantly higher than that of previous biomarkers, which can guide treatment and predict treatment response, and reduce the frequency and risk of invasive examinations.
Smart Images
Figure CN122016987A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical testing and diagnostic technology, specifically relating to the application of an ASGPR1-binding IgA in the diagnosis and treatment efficacy prediction of IgA nephropathy. Background Technology
[0002] IgA nephropathy, one of the most common primary glomerular diseases worldwide, accounts for nearly half of all primary glomerulonephritis diagnosed by renal biopsy in my country. It commonly affects young and middle-aged adults, has a chronic progression, and a poor prognosis; 50% of patients develop uremia after 12.4 years, imposing a severe health and economic burden on families and society. The 2025 updated KDIGO global guidelines for improving glomerular disease state that renal biopsy remains the gold standard for diagnosing IgA nephropathy. However, it is an invasive procedure, performed only in hospitals with renal biopsy technology and renal pathology diagnostic capabilities. Hospitalization is required, and there is a certain risk of bleeding, limiting the early diagnosis of IgA nephropathy. More importantly, IgA nephropathy is chronically progressive, and disease fluctuations are common during follow-up treatment. Renal biopsy cannot be routinely performed repeatedly to guide clinical treatment. Proteinuria level is an important indicator for assessing the prognosis of IgA nephropathy in clinical practice. However, proteinuria is a relatively slow indicator of the severity of nephropathy, and proteinuria level is a common feature of all primary and secondary glomerular diseases, so it does not have the specificity for disease diagnosis.
[0003] The development of non-invasive biomarkers has been a research hotspot in IgA nephropathy. Some researchers have discovered high levels of galactose-deficient IgA1 (GdIgA1) in the circulation of IgA nephropathy patients, which can be measured using a lectinase-mediated immunosorbent assay (ELISA). However, validation at other centers revealed that, firstly, the lectin-based ELISA method is unstable and exhibits high heterogeneity. Secondly, studies in Asian populations have shown limited diagnostic efficacy of GdIgA1 in differentiating IgA nephropathy patients from healthy individuals or other kidney disease controls, with an area under the ROC curve not exceeding 0.8. Furthermore, meta-analysis results indicate significant heterogeneity in GdIgA1 as a biomarker for differentiating IgA, healthy individuals, and other kidney disease controls across different centers. To address the stability of plasma GdIgA1 detection, some researchers have screened antibodies that can identify galactose deficiency in the GdIgA1 hinge region and developed antibody-based ELISA kits for GdIgA1 detection. However, the problems of high heterogeneity in research results, low specificity as a diagnostic indicator, and poor predictive ability for disease prognosis remain unresolved.
[0004] In recent years, commonly used pathogenic IgA-related detection indicators in the research field include IgA-IgG complexes, CD89-captured polypIgA (CD89 pIgA), secretory IgA (sIgA), and dimer IgA (Dimer-IgA). However, these indicators all exhibit low areas under the ROC curve, indicating poor sensitivity and specificity, and thus fail to meet the requirements for biomarkers. Currently, reliable biomarkers for diagnosing IgA nephropathy, assessing changes in kidney disease progression, and predicting prognosis are still lacking.
[0005] Therefore, there is a need for a diagnostic method that is stable, has high diagnostic efficacy, and can predict the severity of disease to address the aforementioned technical challenges. Summary of the Invention
[0006] This invention discloses the following technical solution: the application of ASGPR1-binding IgA (ASGPR1-IgA) as a biomarker for the diagnosis and treatment efficacy prediction of IgA nephropathy.
[0007] The present invention also discloses an enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, wherein the ELISA kit is an ASGPR1 binding IgA ELISA kit.
[0008] Preferably, the enzyme-linked immunosorbent assay kit includes an HRP-labeled IgA antibody.
[0009] Preferably, the enzyme-linked immunosorbent assay kit includes: IgA standard, washing solution, chromogenic substrate and stop solution.
[0010] Preferably, the ASGPR1 comprises the extracellular region of the ASGPR1 protein or an ASGPR1 derivative. ASGPR1 with a His tag is recombinantly expressed in HEK293 cells by transfection with the pcDNA3 plasmid, and the ASGPR1 protein is purified by nickel column affinity chromatography. The recombinant protein used in this invention has the amino acid sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No. 2.
[0011] This invention also discloses a method for detecting IgA nephropathy, which uses the above-mentioned enzyme-linked immunosorbent assay (ELISA) kit and includes the following steps: Step 1: Take an ELISA plate coated with the functional protein ASGPR1, add the plasma to be tested, and incubate. Step 2: After completing the incubation in Step 1, wash the plate with washing buffer and then add HRP-labeled IgA antibody for incubation. Step 3: After completing the incubation in step 2, perform color development, termination, and value reading; Step 4: Perform steps 1 to 3 using IgA standards of different concentrations to create a standard curve; Step 5: Calculate the level of IgA in the plasma to be tested that can bind to the functional protein ASGPR1 based on the standard curve prepared in Step 4. Step 6: Compare the levels of IgA that can bind to the functional protein ASGPR1 in the plasma of the tested patients and healthy or disease controls. Use the ASGPR1-binding IgA value of the plasma samples from healthy or disease controls as a control. Select the optimal cutoff value based on the maximum Youden index of the ROC curve. Values greater than or equal to this cutoff value indicate IgA nephropathy patients, while values less than this cutoff value indicate healthy or disease controls. Sensitivity and specificity are calculated based on the area under the curve (AUC).
[0012] Preferably, step 1 specifically includes: Plate coating: 2.0 ug / ml recombinant ASGPR1 100 ng, incubated overnight at pH 9.6 in bicarbonate coating buffer at 4°C; Washing: 350 μl / well of 0.1% TBST, wash 3 times; Blocking: 150 μl / well of 1% BSA / TBST, incubated at 37°C for 1 h; Washing: 350 μl / well of 0.1% TBST, wash 3 times.
[0013] Preferably, step 2 specifically includes: Sample loading: Dilute the test plasma 1:800 with 0.1% BSA / Tris-Ca solution. The standard is plasma from patients with high test values, serially diluted 1:100. Load 50 μL / well and incubate at 37°C for 1 h. Wash plate: Wash 350 μL / well with 0.1% TBST, 4 times. Detection antibody: Goat anti-human IgA HRP-labeled antibody 1:15000 diluted. Load 50 μL / well and incubate at 37°C for 1 h. Wash plate: Wash 350 μL / well with 0.1% TBST, 4 times.
[0014] Preferably, step 3 specifically includes: color development: TMB color development solution, 50 μL / well, color development at room temperature in the dark for 25 min, OD450 nm absorbance value.
[0015] The beneficial effects of this invention are: 1. This invention, based on IgA glycosylation mass spectrometry analysis, reveals that polyIgA exhibits more significant loss of sialic acid and galactose, exposing more GalNAc. Therefore, this invention provides the theoretical basis for ASGPR1 binding to IgA as a diagnostic tool for IgA nephropathy.
[0016] 2. This invention establishes a reliable and stable ELISA method for cyclic ASGPR1 combined with IgA.
[0017] 3. This invention improves the diagnostic efficacy of IgA nephropathy. Compared with other previous biomarkers, ASGPR1 binding to IgA has greater diagnostic efficacy.
[0018] 4. This invention can guide the treatment of IgA nephropathy. A decrease in ASGPR1-IgA levels indicates that clinical treatment is effective. Therefore, this invention can guide the treatment of IgA nephropathy. Attached Figure Description
[0019] Figure 1 This is a comparison diagram of O-glycosylation mass spectrometry analysis of purified polyIgA according to the present invention; Figure 2 This is a flowchart illustrating the steps of the ELISA method for detecting plasma ASGPR1-binding IgA according to the present invention. Figure 3 The recombinant ASGPR1 of this invention has a higher affinity for purified polyIgA than for monomeric IgA (see diagram). Figure 4 A graph showing the ASGPR1-binding IgA levels of the present invention detected in patients with IgA nephropathy, controls of other kidney diseases, and healthy controls. Figure 5 The area under the ROC curve analysis of the ASGPR1-binding IgA in this invention to distinguish between IgA nephropathy and healthy individuals; Figure 6 The area under the ROC curve analysis of the ASGPR1-binding IgA in this invention to distinguish IgA nephropathy from other nephropathy controls; Figure 7 This is a sequence of serum samples from IgA nephropathy patients for ASGPR1-IgA detection, as presented in this invention.
[0020] In the figure, IgAN represents IgA nephropathy, IgAMM represents IgA myeloma, HC represents healthy controls, and DC represents controls for other kidney diseases. Detailed Implementation
[0021] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0022] like Figures 1-7As shown in this embodiment, the desialylated glycoprotein receptor (ASGPR) is a receptor mainly expressed on the surface of hepatocytes. The main function of ASGPR is to clear desialylated glycoproteins from the blood. IgA is one of the main ligands of ASGPR. ASGPR can specifically recognize and bind to glycoproteins with terminal sialic acid-deficient galactose (Gal) or N-acetylgalactosamine (GalNAc), which is one of the main ways of clearing circulating IgA. Some patients with clinical cirrhosis also have IgA nephropathy. Previous studies in the field of cirrhosis have found that ASGPR can bind to IgA in vitro, and its binding ability to polymeric IgA is stronger than that to monomeric IgA.
[0023] This embodiment, through glycosylation mass spectrometry analysis of circulating polyIgA from IgA nephropathy patients, healthy individuals, and disease controls, shows that the sialic acid (SA) deficiency in polyIgA1 molecules from IgA nephropathy patients is significantly higher than that in healthy individuals and disease controls, which can lead to more GalNAc or Gal exposure. This provides the theoretical basis for establishing the detection of ASGPR1-bound polyIgA in vitro.
[0024] This embodiment, through mass spectrometry analysis of purified polyIgA molecules' O-glycans, determined that the degree of polyIgA sialic acid deficiency was significantly higher than that in healthy individuals and disease controls. Figure 1 As shown, O-glycosylation mass spectrometry analysis of purified polyIgA indicates that polyIgA from patients with IgA nephropathy has a more significant deficiency of sialic acid.
[0025] This embodiment establishes an ELISA method for detecting ASGPR1-binding IgA (ASGPR1-IgA) in serum in vitro. Figure 2 This study verified that recombinant ASGPR1 has a higher affinity for purified polymeric IgA compared to purified monomeric IgA, such as... Figure 3 As shown, the ELISA method verified that ASGPR1 binds to purified polyIgA from patients with IgA nephropathy, and that IgA binding capacity in the plasma of patients with IgA nephropathy is higher.
[0026] Currently, the established ASGPR1-binding IgA in vitro ELISA detection method has been used to detect IgA nephropathy in 100 patients diagnosed by central renal biopsy, 85 controls with other kidney diseases (including membranous nephropathy, minimal change disease, lupus nephritis, focal segmental glomerulosclerosis, ANCA-associated vasculitis, etc.), and 30 healthy controls. Figure 4 As shown, the average level of ASGPR1-IgA in the plasma of patients with IgA nephropathy, controls with other kidney diseases, and healthy individuals was detected by ELISA. The circulating ASGPR1-IgA level in patients with IgA nephropathy was significantly higher than that in controls with other kidney diseases and healthy individuals.
[0027] Regarding diagnostic efficacy, compared with healthy controls, the area under the ROC curve (AUC = 0.852, 95% CI: 0.762–0.943) of ASGPR1-IgA was significantly higher than that of previously detected antibody-based ELISA assays: Gd-IgA1 (AUC = 0.744, 95% CI: 0.624–0.864), CD89-captured polypIgA (CD89 pIgA) (AUC = 0.837, 95% CI: 0.757–0.916), IgA-IgG complex (AUC = 0.725, 95% CI: 0.616–0.834), Dimer-IgA (AUC = 0.807, 95% CI: 0.708–0.906), and sIgA (AUC = 0.850, 95% CI: 0.850). 0.773-0.927), total IgA (AUC = 0.773, 95% CI: 0.668-0.879), details as follows: Figure 5 As shown in Table 1, Table 1 is a table of ROC curve analysis of ASGPR1-binding IgA and other indicators in patients with IgA nephropathy and healthy individuals. As can be seen from Table 1, ASGPR1-binding IgA has a significantly higher area under the ROC curve than other biomarkers in distinguishing between IgA nephropathy and healthy individuals. The combined model of all biomarkers has the best diagnostic efficacy.
[0028]
[0029] Figure 5 The area under the ROC curve analysis distinguishing IgA nephropathy from healthy individuals showed that ASGPR1-binding IgA had a higher area under the curve (AUC) than other biomarkers. Using the ASGPR1-binding IgA values of plasma samples from healthy controls as a control, ROC curve analysis of ASGPR1-binding IgA values in plasma samples from IgA nephropathy patients was performed using SPSS 27.0.1 software. Figure 5 The area under the curve (AUC) is 0.852. Based on the Youden index (sensitivity + specificity - 1) reaching its maximum (sensitivity 90%, specificity 66.7%, Youden index 0.567), the optimal cutoff value was selected as 58.47. Therefore, an ASGPR1-binding IgA value greater than or equal to 58.47 indicates IgA nephropathy (positive), while an ASGPR1-binding IgA value less than 58.47 indicates healthy individuals (negative).
[0030] Compared to other kidney diseases, ASGPR1-IgA remains superior to the other previously used diagnostic indicators, with an AUC of 0.725. Figure 6Table 2 shows the ROC curve analysis of ASGPR1-binding IgA and other indicators in patients with IgA nephropathy and other kidney diseases. As can be seen from Table 2, ASGPR1-binding IgA significantly increased the area under the ROC curve compared to other biomarkers in distinguishing between IgA nephropathy and controls with other kidney diseases. The combined model of all biomarkers showed the best diagnostic efficacy. p All values reached statistical significance.
[0031]
[0032] Figure 6 ROC curve analysis distinguishing IgA nephropathy from controls with other nephropathy showed that ASGPR1-binding IgA had a higher AUC than other biomarkers. Using the ASGPR1-binding IgA value in plasma samples from nephropathy controls (negative), ROC curve analysis was performed on the ASGPR1-binding IgA values in plasma samples from IgA nephropathy patients using SPSS 27.0.1 software. Figure 6 The area under the curve (AUC) is 0.725. Based on the maximum Youden index (sensitivity + specificity - 1) (sensitivity 57%, specificity 77.6%, Youden index 0.346), the optimal cutoff value was selected as 110.03. Therefore, patients with ASGPR1-binding IgA values greater than or equal to 110.03 were classified as having IgA nephropathy (positive), and patients with ASGPR1-binding IgA values less than 110.03 were classified as having non-IgA nephropathy (negative).
[0033] After combining other previously identified biomarkers, and using the combined diagnostic indicators from plasma samples of healthy controls as a control, ROC curve analysis was performed on the combined diagnostic indicators from plasma samples of IgA nephropathy patients using SPSS 27.0.1 software. The area under the curve (AUC) was 0.916, as shown in the figure. Based on the maximum Youden index (sensitivity + specificity - 1) (sensitivity 88.2%, specificity 81.5%, Youden index 0.697), the optimal cutoff value of 0.723 was selected. Therefore, a combined diagnostic indicator value greater than or equal to 0.723 was considered IgA nephropathy (positive), and a combined diagnostic indicator value less than 0.723 was considered healthy (negative). Using the combined diagnostic indicators from plasma samples of other kidney disease controls as a control, ROC curve analysis was performed on the combined diagnostic indicators from plasma samples of IgA nephropathy patients using SPSS 27.0.1 software. The area under the curve (AUC) was 0.835, as shown in the figure. The optimal cutoff value was 0.544 when the Youden index (sensitivity + specificity - 1) was at its maximum (sensitivity was 81.7%, specificity was 75.8%, and Youden index was 0.575). Based on this, patients with a combined diagnostic index greater than or equal to 0.544 were classified as having IgA nephropathy (positive), while patients with a combined diagnostic index less than 0.544 were classified as having other types of kidney disease (negative).
[0034] This invention further analyzed serum ASGPR1-IgA sequences from 19 patients with IgA nephropathy treated with budesonide enteric-coated capsules. The results showed that ASGPR1-IgA levels decreased as proteinuria decreased after treatment, suggesting a predictive effect on treatment response. Figure 7 As shown in the longitudinal cohort of IgA nephropathy, ASGPR1-binding IgA levels decreased as IgA nephropathy patients recovered from treatment. Therefore, ASGPR1-binding IgA levels, as a biomarker, will have significant clinical guiding value for the diagnosis and treatment of IgA nephropathy.
[0035] In summary, this invention discloses the significant value of ASGPR1-binding IgA in the diagnosis and prediction of treatment efficacy in IgA nephropathy. Based on the glycosylation characteristics of polyIgA revealed by IgA glycosylation mass spectrometry analysis, a stable and reliable ELISA method for detecting circulating ASGPR1-binding IgA was established. This method verified the high affinity between recombinant ASGPR1 and polyIgA. Extensive clinical sample testing confirmed that circulating ASGPR1-IgA levels in IgA nephropathy patients were significantly higher than in healthy individuals and other kidney disease controls. In terms of diagnostic efficacy, the area under the ROC curve for ASGPR1-IgA was significantly higher than that of several previous biomarkers, both compared to healthy controls and other kidney disease controls. The diagnostic efficacy was further improved when combined with other biomarkers. Furthermore, analysis of serum samples from treated IgA nephropathy patients revealed that ASGPR1-IgA levels decreased as proteinuria levels decreased after treatment, suggesting its predictive role in treatment response.
[0036] This research provides a new and effective biomarker for the early diagnosis, disease assessment, treatment guidance, and prognosis prediction of IgA nephropathy. It is expected to improve the current situation where IgA nephropathy diagnosis relies on invasive renal biopsies and lacks reliable biomarkers, reducing the frequency of renal biopsies and the risks of bleeding. It will also provide important reference for clinicians to develop personalized treatment plans, thereby improving the treatment effect of IgA nephropathy and reducing the health and economic burden on patients' families and society. In the future, further expansion of the sample size for multi-center clinical validation can be conducted to further investigate the diagnostic and predictive value of ASGPR1-binding IgA in different ethnic and geographical populations, and to explore its combined application with other clinical indicators to better leverage its role in the diagnosis and treatment of IgA nephropathy.
[0037] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.
Claims
1. Application of ASGPR1-binding IgA as a biomarker for the diagnosis and predictive efficacy of IgA nephropathy.
2. An enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy, characterized in that, The enzyme-linked immunosorbent assay (ELISA) kit is an ASGPR1-IgA binding ELISA kit.
3. The enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy according to claim 2, characterized in that, The enzyme-linked immunosorbent assay kit includes an HRP-labeled IgA antibody.
4. The enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy according to claim 2, characterized in that, The enzyme-linked immunosorbent assay kit includes: IgA standard, washing solution, chromogenic substrate and stop solution.
5. An enzyme-linked immunosorbent assay (ELISA) kit for the auxiliary diagnosis of IgA nephropathy according to claim 2, characterized in that, The ASGPR1 includes the extracellular region of the ASGPR1 protein or an ASGPR1 derivative.
6. A method for detecting IgA nephropathy, characterized in that, The detection method uses the enzyme-linked immunosorbent assay kit for the auxiliary diagnosis of IgA nephropathy as described in any one of claims 2 to 5, and the detection method includes the following steps: Step 1: Take an ELISA plate coated with the functional protein ASGPR1, add the plasma to be tested, and incubate. Step 2: After completing the incubation in Step 1, wash the plate with washing buffer and then add HRP-labeled IgA antibody for incubation. Step 3: After completing the incubation in step 2, perform color development, termination, and value reading; Step 4: Perform steps 1 to 3 using IgA standards of different concentrations to create a standard curve; Step 5: Calculate the level of IgA in the plasma to be tested that can bind to the functional protein ASGPR1 based on the standard curve prepared in Step 4. Step 6: Compare the levels of IgA that can bind to the functional protein ASGPR1 in the plasma of the test patients and healthy or disease control patients; use the ASGPR1-binding IgA value of the plasma samples of healthy or disease control patients as a control, and select the optimal cutoff value when the Youden index of the ROC curve is maximized. Patients with IgA nephropathy are those with values greater than or equal to this cutoff value, and those with values less than this cutoff value are healthy or disease control patients.
7. The method for detecting IgA nephropathy according to claim 6, characterized in that, Step 1 specifically includes: Plate coating: 2.0 ug / ml recombinant ASGPR1 100 ng, incubated overnight at pH 9.6 in bicarbonate plate coating solution at 4°C; Washing: 0.1% TBST, 350ul / well, 3 washes; Blocking: 1% BSA / TBST 150ul / well, incubate at 37°C for 1 hour; Washing: Wash 350ul / well with 0.1% TBST, 3 times.
8. The method for detecting IgA nephropathy according to claim 6, characterized in that, Step 2 specifically includes: Sample addition: Dilute the plasma to be tested 1:800 with 0.1 BSA / Tris-Ca solution. The standard is a 1:100 serial dilution of plasma from patients with high test values. Add 50 μL / well and incubate at 37°C for 1 h. Washing: Wash 350ul / well with 0.1% TBST, 4 times; Antibody detection: Goat anti-human IgA HRP-labeled antibody diluted 1:15000, with a sample volume of 50 μL / well, and incubated at 37°C for 1 h; Washing: Wash 350ul / well with 0.1% TBST, 4 times.
9. The method for detecting IgA nephropathy according to claim 6, characterized in that, Step 3 specifically includes: color development: TMB color development solution, 50ul / well, color development at room temperature in the dark for 15-25min, OD450nm absorbance value.