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CN-122016987-A - Application of ASGPR 1-binding IgA in IgA nephropathy diagnosis and treatment effect prediction markers

CN122016987ACN 122016987 ACN122016987 ACN 122016987ACN-122016987-A

Abstract

The invention belongs to the technical field of medical detection and diagnosis, relates to application of ASGPR 1-combined IgA (ASGPR-IgA) in IgA nephropathy diagnosis and treatment effect prediction markers, and discloses important value of ASGPR 1-combined IgA in IgA nephropathy diagnosis and treatment effect prediction. Based on the obvious glycosylation characteristic change of the multimeric IgA sialic acid deletion revealed by the analysis of IgA glycosylation mass spectrum, a stable and reliable ELISA detection method for circulating ASGPR 1-binding IgA is established. The method verifies the high affinity of recombinant ASGPR1 and multimeric IgA, and through a large number of clinical sample detection, the circulating ASGPR1-IgA of IgA nephropathy patients is obviously higher than that of healthy people and other nephropathy controls, and the diagnosis efficacy is superior to that of other biomarkers in the past.

Inventors

  • XIE XINFANG
  • YUAN XIAOHAN
  • LV JICHENG

Assignees

  • 西安交通大学医学院第一附属医院

Dates

Publication Date
20260512
Application Date
20260209

Claims (9)

  1. Application of ASGPR 1-binding IgA in IgA nephropathy diagnosis and treatment effect prediction markers.
  2. 2. An enzyme-linked immunosorbent assay kit for assisting in diagnosing IgA nephropathy, characterized in that the enzyme-linked immunosorbent assay kit is an ASGPR 1-to-IgA ELISA kit.
  3. 3. An enzyme-linked immunosorbent assay kit for aiding diagnosis of IgA nephropathy according to claim 2, wherein the enzyme-linked immunosorbent assay kit comprises an HRP-labeled IgA antibody.
  4. 4. The ELISA kit for aiding diagnosis of IgA nephropathy according to claim 2, wherein the ELISA kit comprises an IgA standard, a washing solution, a chromogenic substrate and a stop solution.
  5. 5. An enzyme-linked immunosorbent assay kit for aiding diagnosis of IgA nephropathy according to claim 2, wherein the ASGPR1 comprises an extracellular region of an ASGPR1 protein or an ASGPR1 derivative.
  6. 6. An IgA nephropathy detection method, characterized in that the detection method employs the enzyme-linked immunosorbent kit for aiding diagnosis of IgA nephropathy as claimed in any one of claims 2 to 5, comprising the steps of: Step 1, taking an ELISA plate coated with a functional protein ASGPR1, and adding plasma to be detected for incubation; Step 2, after the incubation in the step 1 is completed, washing the plate by adopting a washing liquid, and then adding an HRP-marked IgA antibody for incubation; step 3, after the incubation in the step 2 is completed, color development, termination and value reading are carried out; step 4, adopting IgA standard substances with different concentrations to perform the operations from the step 1 to the step 3, and manufacturing a standard curve; step 5, calculating to obtain the IgA level which can be combined with the functional protein ASGPR1 in the plasma to be detected according to the standard curve prepared in the step 4; And 6, comparing the IgA level which can be combined with the functional protein ASGPR1 in the plasma of the patient to be tested and the healthy control or disease control, taking the ASGPR1 combined IgA value of the plasma sample of the healthy control or disease control as a control, selecting an optimal limit value according to the condition that the ROC curve about step index is maximum, wherein the IgA nephropathy patient is greater than or equal to the limit value, and the healthy or disease control is smaller than the limit value.
  7. 7. The IgA nephropathy detection method according to claim 6, wherein the step 1 specifically comprises: Plating 2.0ug/ml recombinant ASGPR1 100ng incubated at ph=9.6 bicarbonate plating 4-degree overnight; washing the plates, namely, 0.1 percent TBST washing the plates for 350 ul/hole for 3 times; Blocking 1% BSA/TBST 150 ul/well, incubation at 37℃for 1h; Wash plate 0.1% TBST plate 350 ul/well, 3 washes.
  8. 8. The IgA nephropathy detection method according to claim 6, wherein the step 2 specifically comprises: sample addition, namely diluting the plasma to be detected 1:800 by 0.1BSA/Tris-Ca liquid, diluting the plasma of a patient with higher detection value by 1:100 times as a standard substance, and incubating for 1h at 37 ℃ by 50 ul/hole of sample addition volume; washing the plates, namely, 0.1% TBST washing the plates for 350 ul/hole, and washing for 4 times; The detection antibody is diluted 1:15000 by goat anti-human IgA HRP labeled antibody, the sample adding volume is 50 ul/hole, and the incubation is carried out for 1h at 37 ℃; wash plate 0.1% TBST plate 350 ul/well, 4 washes.
  9. 9. The IgA nephropathy detection method according to claim 6, wherein the step 3 specifically comprises the steps of developing TMB color development liquid, developing 50 ul/hole, developing at room temperature in a dark place for 15-25min, and absorbing the absorbance value at OD450 nm.

Description

Application of ASGPR 1-binding IgA in IgA nephropathy diagnosis and treatment effect prediction markers Technical Field The invention belongs to the technical field of medical detection and diagnosis, and particularly relates to application of ASGPR 1-combined IgA in IgA nephropathy diagnosis and treatment effect prediction markers. Background IgA nephropathy is one of the most common primary glomerular diseases worldwide, accounting for approximately half of the primary glomerulonephritis diagnosed by renal biopsy in China. The disease is well developed in young and middle-aged people, chronic progression is poor in prognosis, 50% of patients enter uremia after 12.4 years, and serious sanitary and economic burden is brought to families and society. The global guidelines for improving glomerular disease, updated in 2025, suggest that kidney biopsy is still the gold standard for diagnosis of IgA nephropathy, but is an invasive procedure, which is only carried out in hospitals that develop kidney biopsy techniques and have kidney pathology diagnosis capabilities, require hospitalization and have a certain bleeding risk, and limit early diagnosis of IgA nephropathy. More importantly, igA nephropathy progresses chronically, and condition fluctuations often occur in follow-up treatment, while kidney puncture biopsies cannot be used as routine repeated procedures for guiding clinical treatment. Proteinuria levels are currently an important clinical indicator for assessing IgA nephropathy kidney prognosis, but the severity of proteinuria response nephropathy is relatively slow, and proteinuria levels are common characteristics of all primary and secondary glomerular lesions, and do not have disease diagnosis specificity. Non-invasive biomarker development has been a research hotspot for IgA nephropathy. The higher levels of galactose-deficient IgA1 (GdIgA 1) found in the circulation of IgA nephropathy patients by students can be determined by a lectin enzyme-mediated coupled immunosorbent assay. However, in other central validation processes, the lectin-based ELISA method was found to be unstable and heterogeneous first. Secondly, asian population researches find that the GdIgA index has limited diagnosis efficacy in distinguishing IgA nephropathy patients from healthy people or other nephropathy controls, the area under the ROC curve is not more than 0.8, and the meta-analysis result shows that GdIgA is used as a biomarker to identify IgAN, healthy people and other nephropathy controls with great heterogeneity in different center results. In order to solve the stability of plasma GdIgA1 detection, students screen antibodies capable of recognizing galactose defects in GdIgA1 hinge region, and establish an ELISA kit for detecting GdIgA1 based on an antibody method, but the problems of large heterogeneity of research results and low specificity as a diagnosis index are still not solved, and the disease prognosis prediction capability is poor. In recent years, detection indicators related to pathogenic IgA commonly used in the scientific field include IgA-IgG complexes, CD89 captured multimers pIgA (CD 89 pIgA), secretory IgA (sIgA) and dimeric IgA (Dimer-IgA). However, all the detection indexes are shown that the area value under the ROC curve is lower, the sensitivity and the specificity are poorer, and the biomarker requirements cannot be met. There is still a lack of reliable biomarkers for diagnosis, assessment of renal disease changes and prognosis prediction of IgA nephropathy. Therefore, there is a need for a biomarker that is stable in diagnostic methods, has high diagnostic efficacy, and can predict disease severity to solve the above-mentioned problems. Disclosure of Invention The invention discloses a technical scheme that application of ASGPR 1-combined IgA (ASGPR 1-IgA) in IgA nephropathy diagnosis and treatment effect prediction markers. The invention also discloses an ELISA kit for assisting in diagnosing IgA nephropathy, which is an ELISA kit for combining ASGPR1 with IgA. Preferably, the enzyme-linked immunosorbent assay kit comprises an HRP-labeled IgA antibody. Preferably, the ELISA kit comprises an IgA standard, a washing solution, a chromogenic substrate and a stop solution. Preferably, the ASGPR1 comprises an extracellular region of ASGPR1 protein or an ASGPR1 derivative. ASGPR1 with His tag was expressed recombinantly by pcDNA3 plasmid transfected into HEK293 cells and ASGPR1 protein was purified by nickel column affinity chromatography. The amino acid sequence of the recombinant protein used in the invention is shown as SEQ ID No.1, and the nucleotide sequence is shown as SEQ ID No. 2. The invention also discloses an IgA nephropathy detection method, which adopts the ELISA kit, and comprises the following steps: Step 1, taking an ELISA plate coated with a functional protein ASGPR1, and adding plasma to be detected for incubation; Step 2, after the incubation in the step 1 is completed, washin