HCV NS3 antibody detection method based on MIX antigen coating

A detection method and antigen technology, applied in the field of biotechnology diagnosis, can solve the problem of not being able to detect antibodies as effectively, and achieve the effect of reducing missed detection and improving the detection rate

Inactive Publication Date: 2019-03-08
WUXI PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In mainland China, genotype 1 is the main type, but there are still mixed infections of other genotypes, so the NS3 antigen with a specific genotype as type 1 cannot detect antibodies of other genotypes equally effectively

Method used

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  • HCV NS3 antibody detection method based on MIX antigen coating
  • HCV NS3 antibody detection method based on MIX antigen coating
  • HCV NS3 antibody detection method based on MIX antigen coating

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] HCV NS3 antibody detection program optimization

[0045] 1. Selection of the optimal dilution of NS3MIX coating antigen and enzyme-labeled antibody

[0046] 1. Method: Use the square array titration method to coat the purified MIX antigen containing HCV NS3 (type 1) and NS3 (type 6) in multiple dilutions, and at the same time, detect the HRP-labeled goat anti-human IgG For doubling dilution, the dilution ratios of both are from 1:200 to 1:3200, and 2 negative samples and 2 positive samples are detected respectively, and the sample volume is 1 μl, and a blank control well is set up. The specific operation steps are as follows, Finally select the positive serum OD 450 Value and Negative Serum OD 450 The dilution with the highest ratio of values ​​is the optimal dilution.

[0047] 2. Specific steps: Dilute the NS3MIX antigen with 0.01mol / L PBS (pH 7.2) and coat the microplate, 100μl / well, put it in a refrigerator at 4°C overnight; wash 3 times with 1×PBST solution the n...

Embodiment 2

[0078] Repeatability and Stability Test of HCV NS3 Antibody Detection

[0079] 1. Repetitive test

[0080]1. Method: Get 3 positive specimens, measure 10 times simultaneously, calculate intra-assay coefficient of variation (CV), measure once a day for the same 3 specimens and measure continuously for 5 days, calculate inter-assay coefficient of variation.

[0081] 2. Specific steps: the detailed operation steps and methods are the same as items 1 and 2 in Example 1.

[0082] 3. Results: 3 positive serum samples were measured 10 times at the same time period, and the intra-assay coefficient of variation was calculated; the same 3 positive serum samples were continuously measured 5 times, once a day, and the inter-assay coefficient of variation was calculated; the result coefficients of variation were all less than 10%, showing that the repeatability of the method is good (shown in the table below).

[0083] Repeatability test results (CV)

[0084]

[0085] 2. Stability te...

Embodiment 3

[0093] Sensitivity and Specificity Test of HCV NS3 Antibody Detection

[0094] 1. Methods and steps

[0095] (1) Detect 90 anti-HCV antibody positive sera.

[0096] (2) HCV antigen neutralization test: take 10 anti-HCV antibody positive sera and add HCV recombinant MIX antigen respectively, and incubate at 37°C for 1 hour; take the same 10 positive sera as the control group, but without adding MIX antigen, do the same Incubate at 37°C for 1 hour; then test the samples with the established detection method respectively.

[0097] (3) 6 anti-HAV antibody positive, 9 anti-HBV antibody positive, and 20 anti-HEV antibody positive serum samples were tested respectively.

[0098] (4) Detect 200 serum samples from healthy physical examination and 20 negative quality control serum samples from clinical examination centers.

[0099] 2. Results

[0100] (1) 90 anti-HCV antibody positive serum specimens were tested, and as a result, 65 were positive for HCV NS3 antibody, and the positi...

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PUM

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Abstract

The invention proposes an HCV NS3 abzyme immunoassay method based on MIX antigen coating. The HCV NS3 abzyme immunoassay method includes diluting HCV NS3 (type 1 and type 6) MIX antigens with 0.01mol / L of PBS (pH 7.2), coating a microporous board (100 microliter / pore) with the antigens, putting the board in a refrigerator at the temperature of 4 DEG C overnight, washing the board 3 times with 1*PBST solutions the next day, performing drying and sealing, and storing the board at the temperature of 4 DEG C for standby use; adding diluted enzyme-labeled secondary antibodies (goat anti-human IgG)into enzyme-labeled pores (100 microliter / pore), adding 1 microliter of serum specimens, dividing the serum specimens into a blank control group, a negative control group and positive control group, and to-be-tested serum, and performing gentle mixing; performing incubation at the temperature of 37 DEG C for 1 hour. According to the current situation that domestic HCV antibody detection reagents are still not perfect, as well as the characteristics of Chinese native HCV genotype distribution, the HCV NS3 abzyme immunoassay method based on MIX antigen coating is established to increase the detection rate of HCV antibodies, thereby reducing detection omission of HCV-infected persons.

Description

technical field [0001] The invention relates to the technical field of biotechnology diagnosis, in particular to an HCV NS3 antibody detection method based on MIX antigen coating. Background technique [0002] At present, enzyme immunoassay (EIA) is widely used to detect anti-HCV as the diagnostic index of hepatitis C (HC). Currently, blood donors are routinely screened for anti-HCV in China, which greatly reduces the incidence of HC after blood transfusion, but there are still Some blood recipients developed post-transfusion HC, which proves that the current domestic HCV detection reagents are not perfect; HCV is prone to mutation, and its gene sequence is highly heterogeneous. At present, HCV is divided into 6 genotypes and more than 20 genotypes. Different genotypes of HCV are widely distributed in different regions of the world. In Asia, HCV types 1, 2, and 6 are distributed, and in China, HCV is mainly type 1b. However, mixed infection is significantly higher than that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/576
CPCG01N33/5767G01N33/6854
Inventor 乔伟振李另另乔简
Owner WUXI PEOPLES HOSPITAL
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