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A detection kit for anti-peptidyl prolyl cis-trans isomerase d-igg antibody

A technology for peptidyl prolyl cis and basal prolyl cis, which is applied in the field of biomedicine, can solve problems such as unreported relationship, blank and the like, and achieves the effects of simple cleaning and separation, easy operation, and reducing the probability of cross infection

Active Publication Date: 2022-05-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there is no report on the relationship between peptidyl-prolyl cis-trans isomerase D and nephrotic syndrome
In the prior art, there is currently no research on the identification of autoimmune nephrotic syndrome by detecting serum anti-peptidyl-prolyl cis-trans isomerase D-IgG antibodies

Method used

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  • A detection kit for anti-peptidyl prolyl cis-trans isomerase d-igg antibody
  • A detection kit for anti-peptidyl prolyl cis-trans isomerase d-igg antibody
  • A detection kit for anti-peptidyl prolyl cis-trans isomerase d-igg antibody

Examples

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Effect test

Embodiment 1

[0036] Example 1 Peptidyl-prolyl cis-trans isomerase D on podocytes is the target antigen for autoantibodies in patients with autoimmune nephrotic syndrome

[0037] (1) Extraction of total protein of glomerular podocytes: culture podocyte strain (MPC5), wash 2-3 times with PBS, and then use a focused ultrasonic instrument (Covaris S220, Gene) in a medium containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitors (#ab65621; Abcam, 1:200 dilution) were fully lysed on ice in the lysis buffer, and then the sample was placed in a centrifuge at 12000g, 4°C for 30min. The supernatant was collected, which was the collected glomerular podocyte total protein. The total protein concentration of collected glomerular podocytes was determined by BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: the total protein of glomerular podocytes was extracted for two-dimensional electrophoresis, then transferred to a nitrocellulose membrane, incubated with serum from...

Embodiment 2

[0038] Example 2 Expression and purification of recombinant peptidyl-prolyl cis-trans isomerase D antigen protein

[0039] The method of genetic engineering is used to use the gene encoding peptidyl-prolyl cis-trans isomerase D protein as a template to carry out PCR amplification, and then construct an expression vector for protein expression. The antigenic protein expressed in the present invention contains the tag peptide of the His tag. The expressed recombinant protein was purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc. Finally, the molecular weight of the recombinant protein peptidyl-prolyl cis-trans isomerase D was identified by SDS-PAGE as 41KDa, the results are shown in figure 2 .

Embodiment 3

[0040]Example 3 The present invention uses an orthogonal experiment design to optimize the reaction conditions of the kit According to the coating concentration of antigen peptidyl-prolyl cis-trans isomerase D (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL four coating Concentration), each reaction time (30min, 45min) and temperature (25°C, 35°C), the optimal dilution of the enzyme-labeled secondary antibody (1:100, 1:500, 1:1000, 1:1500 four dilutions) Choose an orthogonal table for 4 factors, each factor is repeated at 2 levels for standard positive serum and standard negative serum. Select the ratio (P / N) of the highest luminescence value (P) of the positive serum to the lowest luminescence value (N) of the negative serum. The average P / N value of repeated determinations was determined through statistical processing to determine the optimal coating conditions and the optimal dilution of the secondary antibody for orthogonal optimization conditions, which significantly improved ...

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Abstract

The invention provides a detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody. When the detected serum sample is in contact with the target antigen peptidyl-prolyl cis-trans isomerase D (peptidyl prolyl cis-trans isomerase D) polypeptide or its binding fragment, an immune reaction occurs and an antigen-antibody complex is formed. Anti-peptidylprolyl cis-trans isomerase D‑IgG autoantibodies were detected using the antigen-antibody complexes. The present invention identifies the anti-peptidyl prolyl cis-trans isomerase D-IgG antibody for the first time in children with nephrotic syndrome, and provides a detection kit for the antibody. To provide a better detection method for the identification of autoimmune nephrotic syndrome related to anti-peptidyl prolyl cis-trans isomerase D-IgG antibody at home and abroad.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody. Background technique [0002] Primary nephrotic syndrome (PNS) is caused by a variety of factors that increase the permeability of the glomerular basement membrane, and then increase the filtration of plasma protein and lose it in the urine, which leads to a series of pathological changes. clinical syndrome. PNS is a common glomerular disease in children, with an annual incidence of 1.15-16.9 / 100000 according to reports. The pathological type is minimal change nephrotic syndrome (MCNS), which is the most common. According to the response effect of PNS to hormone therapy, it can be divided into steroid-sensitive type (SSNS), steroid-dependent type (SDNS), and steroid-dependent type (SDNS). Drug-resistant type (SRNS). Although the vast majority of children are sensitive to hormone therapy and have a go...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/564G01N33/573G01N33/543G01N21/76
CPCG01N33/564G01N33/573G01N33/54326G01N21/76G01N2333/99G01N2800/24G01N2800/347
Inventor 叶青毛建华张俊峰
Owner ZHEJIANG UNIV
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