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CN-122017252-A - High-sensitivity kit for detecting Gd-IgA in peripheral blood/urine as well as preparation method and application thereof

CN122017252ACN 122017252 ACN122017252 ACN 122017252ACN-122017252-A

Abstract

The invention belongs to the technical field of biological detection, and provides a high-sensitivity kit for detecting Gd-IgA in peripheral blood/urine, and a preparation method and application thereof. The method has high sensitivity, strong specificity, stable and reliable result and simple operation.

Inventors

  • REN XIANQING
  • REN YINGJIE
  • WANG YAN
  • Cang Mengting
  • TANG JINFA
  • ZHANG DAI
  • JIANG LU
  • JIANG YINGYING
  • Yu Diewen
  • HUA LEI
  • Li Liuxu
  • ZHANG MINGLIANG

Assignees

  • 河南中医药大学第一附属医院

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. A high-sensitivity kit for detecting Gd-IgA in peripheral blood/urine is characterized by comprising a Protein A/G fusion Protein pre-coated micro-pore plate, a confining liquid and an anti-Gd-IgA monoclonal antibody.
  2. 2. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 1, wherein the preparation method of the Protein a/G fusion Protein pre-coated microwell plate comprises the steps of: Diluting Protein A/G fusion Protein with coating buffer solution, adding diluted liquid into each well of the micro-porous plate, incubating for 12-16 hours at 4 ℃, then discarding the liquid in the well, washing, and removing residual liquid in the well.
  3. 3. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 2 wherein the coating buffer is 0.05M carbonate buffer.
  4. 4. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 1, wherein the Protein a/G fusion Protein has a coating concentration of 2.0-2.5 μg/mL.
  5. 5. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 1 wherein the anti-Gd-IgA monoclonal antibody is Km55 monoclonal antibody at a concentration of 0.04-0.06 μg/mL.
  6. 6. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 1 wherein the blocking solution is phosphate buffer containing 0.8-1.2 g/100 mL gelatin and 0.8-1.2 g/100 mL bovine serum albumin.
  7. 7. The high sensitivity kit for detecting Gd-IgA in peripheral blood/urine according to claim 1 wherein the blocking solution is phosphate buffer containing 1 g/100 mL gelatin and 1 g/100 mL bovine serum albumin.
  8. 8. A method for preparing a kit according to any one of claims 1 to 7, comprising the steps of: S1, adding Protein A/G fusion Protein into each hole of a micro-pore plate, incubating at 4 ℃, washing, and removing residual liquid in the hole; S2, adding a sealing liquid into the microporous plate, incubating at a constant temperature of 37 ℃, discarding the sealing liquid, washing, and removing residual liquid in the holes; s3, adding an anti-Gd-IgA monoclonal antibody into the micro-porous plate, incubating at 37 ℃, washing, and removing residual liquid in the pores; s4, vacuum sealing the micro-pore plate in an aluminum foil sealing bag, and preserving.
  9. 9. The method of claim 8, wherein the washing is performed with a washing solution comprising phosphate buffer containing 0.05% -0.1% tween-20.
  10. 10. Use of a kit according to any one of claims 1-7 for the preparation of an in vitro test product for aiding diagnosis or monitoring of Gd-IgA-related kidney diseases including IgA nephropathy and allergic purpuric nephritis.

Description

High-sensitivity kit for detecting Gd-IgA in peripheral blood/urine as well as preparation method and application thereof Technical Field The invention belongs to the technical field of biological detection, and relates to a high-sensitivity kit for detecting Gd-IgA in peripheral blood/urine, and a preparation method and application thereof. Background IgA nephropathy is the most common primary glomerulonephritis worldwide, and its diagnostic gold standard is invasive renal aspiration biopsy. Studies show that galactose-deficient IgA1 (Gd-IgA 1 for short) plays a central role in pathogenesis of IgA nephropathy, and Gd-IgA1 level in serum and urine of patients is obviously increased and is closely related to disease activity and progression risk. Therefore, the development of a high-sensitivity and high-specificity Gd-IgA1 detection method has important clinical value for noninvasive auxiliary diagnosis, risk stratification and prognosis monitoring of the disease. Currently, the mainstream method for detecting Gd-IgA1 is an enzyme-linked immunosorbent assay, abbreviated as ELISA. The conventional ELISA method often uses a blocking solution such as 5% skimmed milk powder, but its blocking effect is limited. Conventional ELISA methods also typically employ a means of directly coating the capture antibodies onto a common polystyrene microplate. The method has the obvious defects that firstly, the specificity is poor, the directly coated antibody is easy to be combined with other substances in a sample in a non-specific way due to the fact that the detection target is a special glycosylation epitope on IgA molecules, so that background signals are high, the false positive rate is high, secondly, the sensitivity is limited, the low-concentration Gd-IgA1 in blood or urine can not be accurately quantified due to the fact that the sensitivity is limited, the application of the method in early diagnosis is limited, furthermore, the detection result is unstable, the combination efficiency of the enzyme-labeled antibody is greatly influenced by a coating process, the difference among batches is obvious, and finally, the cost is high, the market depends on an imported kit at present, and the popularization difficulty of clinical application is increased. For example, chinese patent application with publication number of CN 114478787A discloses an anti-Gd-IgA 1 monoclonal antibody and an ELISA kit for IgA nephropathy auxiliary diagnosis, and the human Gd-IgA1 Elisa kit provided by the scheme can well identify Gd-IgA1 standard substances, is linearly related within the range of 1.25-100ng/mL, and does not react with normal IgA1 protein. The kit can be used for diagnosing and distinguishing normal human, non-IgAN nephropathy patients and IgAN nephropathy patients, wherein the serum Gd-IgA1 level of the IgAN nephropathy patients is obviously higher than that of the normal human, non-IgAN nephropathy patients. The sensitivity of the kit for diagnosing Ig AN is 73.33%, the specificity is 83.33% and the AUC is 0.8022 by taking serum Gd-IgA1> 8235 ng/mL as a critical value. However, the kit is only suitable for serum/plasma, does not cover urine samples, and limits the application range of the kit in kidney disease monitoring. Therefore, there is a need in the art for a better performing, lower cost Gd-IgA kit and assay method. Disclosure of Invention The invention provides a high-sensitivity kit for detecting Gd-IgA in peripheral blood/urine, and a preparation method and application thereof. The technical scheme of the invention is realized as follows: Subject 1 A high-sensitivity kit for detecting Gd-IgA in peripheral blood/urine comprises a Protein A/G fusion Protein pre-coated micro-pore plate, a confining liquid and an anti-Gd-IgA monoclonal antibody. Preferably, the kit further comprises an enzyme-labeled anti-human IgA antibody, a human Gd-IgA1 standard, a color development solution and a stop solution. Wherein, the enzyme-labeled anti-human IgA antibody, the human Gd-IgA1 standard substance, the chromogenic liquid and the stop solution are adopted in the detection method process. Preferably, the enzyme-labeled anti-human IgA antibody employs a F (ab') 2 fragment. Preferably, the preparation method of the Protein A/G fusion Protein pre-coated micro-pore plate comprises the following steps: Diluting Protein A/G fusion Protein with coating buffer solution, adding diluted liquid into each well of the micro-porous plate, incubating for 12-16 hours at 4 ℃, then discarding the liquid in the well, washing, and removing residual liquid in the well. Preferably, the coating buffer is a 0.05M carbonate buffer. Preferably, the carbonate buffer is a mixed aqueous solution of sodium carbonate and sodium bicarbonate. Preferably, the Protein A/G fusion Protein has a coating concentration of 2.0-2.5. Mu.g/mL. Preferably, the anti-Gd-IgA monoclonal antibody is Km55 monoclonal antibody, and the concentration of the anti-Gd-IgA monoclonal a