Chemiluminiscence kit for detecting anti-envelope glycoprotein antibody in serum of HCV infected person, and detection method

Abstract

The invention relates to the technical field of biology, and discloses a chemiluminiscence kit for detecting anti-envelope glycoprotein (anti-E1 and anti-E2) antibodies in serum of an HCV infected person. The kit comprises a microwell plate obtained by coating with a recombinant HCV envelope glycoprotein (E1, E2) antigen, and a sample diluent, a negative control, a positive control, an enzyme marker working solution, a substrate solution A, a substrate solution B and a washing solution which are independently packaged, wherein the substrate solution A comprises luminol, o-phenylphenol, 4-imidazole phenol and a carbonic acid buffer solution (CB), and the substrate solution B comprises urea peroxide and a phosphate buffer solution (PB). According to the invention, the kit can be used for carrying out in-vitro qualitative / quantitative determination on anti-E1 and anti-E2 antibodies in serum of an HCV infected person, establishing a detection technology of the anti-E1 and anti-E2 antibodies in the serum of the HCV infected person and carrying out methodological evaluation, and can be applied to screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of the HCV infected person.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a qualitative / quantitative reagent kit for chemiluminescence immunoassay, in particular to a micropore coated with recombinant hepatitis C virus (hepatitis C virus, HCV) envelope glycoprotein (E1, E2) antigen Plate, detect the generated coated antigen-test antibody-enzyme-labeled secondary antibody complex, can perform qualitative / quantitative determination of anti-E1 and anti-E2 antibodies in the serum of HCV-infected patients in vitro, and establish anti-E1, anti-E1 and anti-E2 antibodies in the serum of HCV-infected patients Anti-E2 antibody detection technology and methodological evaluation, the kit can be applied to the screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of hepatitis C virus. Background technique [0002] In 1974, Golafield M first reported non-A, non-B hepatitis after blood transfusion. In 1989, Choo QL, W...

AI Technical Summary

Problems solved by technology

As the treatment of HCV infection enters the pan-gene era, the direct use of recombinant protease inhibitors (Direct Acting Antiviral, DAA) can greatly increase the proportion of patients achieving sustained virological response (>95%), which is expected to change the severe situation of HCV infection threat, However, the current DAA treatment still faces many problems to be solved: (1) "Marginal groups" cannot receive effective treatment, such as injecting drug users, inmates, and gay men, which exacerbate the prevalence of the disease; (2) Standardized DAA treatment After treatment, there are still some patients who do not have sustained virological response or treatment failure, and DAA treatment cannot prevent HCV re-infection; (3) The proportion of medical insurance payment is low and the treatment cost is expensive, and the treatment cost of each course of treatment is tens of thousands to tens of thousands ten thousand RMB

[0005] However, the research on chemiluminescent immunoassay kits and related technologies for the detection of anti-envelope glycoprotein E1 and E2 antibodies is still blank at home and abroad. Some foreign scholars have used recombinant Core, E1, E2, NS3, NS4A, NS4B, NS5A, NS5B protein established immunoblotting method to detect the corresponding protein antibody in serum of HCV infected patients ( M, Melén K, PorkkaP, et al.Hepatitis Cvirus core, NS3, NS4Band NS5A are the majorimmunogenic proteins in humoral immunity in chronic HCV infection [J]. Virol J, 2009, 6:84.), the results show that: in HCV infection There are differences in the positive rates of antibodies for different proteins, which may be related to the different progression of HCV infection, different genotypes, before and after DAA treatment, and the presence of circulating immune complexes in serum.

Due to the low sensitivity of immunoblotting, the conventional kits used for clinical diagnosis of hepatitis C all use HCV nucleocapsid region recombinant protein C-22, nonstructural protein NS3 region recombinant protein C-200 antigen, nonstructural protein Recombinant protein C-200 in the NS4 region and NS5 etc. are used as the fourth-generation reagents for mixing and coating antigens to detect antibodies (Warkad SD, SongKS, PalD, et al. Developments in the HCVScreening Technologies Based on the Detection of Antigens and Antibodies. Sensors (Basel) .2019,19(19):4257.), the methods for detecting antibodies mainly include enzyme-linked immunosorbent assay (ELISA), colloidal gold method, and as the application publication number is CN103018455A disclosed chemiluminescent method to directly label antigen to detect hepatitis C Viral antigen antibody and detection kits, but these kits can only detect mixed antibodies in the serum of HCV-infected patients, and cannot distinguish what kind of protein components are antibodies, and cannot fully reveal the individual HCV protein components in the condition monitoring and prognosis evaluation of HCV-infected patients and epidemiological research; for example, envelope glycoprotein E1 stimulates the body to produce antibodies (anti-E1) and circulating immune complexes (E1-IC). Stage (chronic, cirrhosis, liver cancer), is there any difference before and after antiviral treatment? So far, there have been no relevant reports

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