Improved double-antigen sandwiching immunity detection method

A double-antigen sandwich and antibody technology, applied in the field of immunodetection, can solve the problems of reduced sensitivity, high false positive rate and high background, and achieve the effect of reducing background and false positive rate and improving specificity

Active Publication Date: 2013-06-26
GUANGDONG WESAIL BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, reducing the amount of coated or labeled antigen will inevitably reduce the sensitivity of the detection, and it is impossible to increase the purity of the antigen to make the purity of the antigen 100%. The quality of the kit material is not a factor that can be adjusted at will.
Therefore, when the double-antigen sandwich method detects the target antibody, the background is too high and the false positive rate is always a problem that is difficult to solve.

Method used

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  • Improved double-antigen sandwiching immunity detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 HCV double-antigen sandwich method gold standard test strip with anti-human IgG polyclonal antibody added to the sample pad

[0030] 1.1 Preparation of HCV coating antigen

[0031]PCR amplifies the DNA segment corresponding to the NS3 part of the hepatitis C virus gene such as SEQ ID NO.1 (belonging to a part of the NS3 protein), and its upstream primer is as shown in SEQ ID NO.2, with a BamHI site, and the downstream primer As shown in SEQ ID NO.3, it has a HindIII site. After the PCR fragment was recovered, it was digested with BamHI and HindIII, and connected to the PQE30 vector (Qiagen Company) digested with BamHI and HindIII to obtain the positive recombinant plasmid PQE30-NS3. The plasmid pQE30-NS3 was transformed into ER2566 bacteria (New England Biolabs, USA), and 500ml of LB medium containing 100ug / ml ampicillin sodium (Shanghai Sangon Bioengineering Technology Service Co., Ltd., catalog number A0339) was used for shaking culture at 37°C until OD600=...

Embodiment 2

[0058] Example 2 HCV double-antigen sandwich method gold standard test strip with anti-human IgG polyclonal antibody added to the marker pad

[0059] Refer to Examples 1.1, 1.2, 1.3, and 1.4 to prepare HCV coating antigen, HCV labeled antigen, colloidal gold, and labeled antigen-colloidal gold complex.

[0060] 2.5 Assembly of test strips

[0061] Dilute the mouse anti-human IgG polyclonal antibody to 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0mg / ml with PBS buffer, soak glass fiber (Watman company) respectively, -50 ℃ vacuum freeze-drying for 6 hours and then stored at 4 ℃. PBS without mouse anti-human IgG polyclonal antibody was used as a control.

[0062] Dilute the labeled antigen-colloidal gold complex 10 times with colloidal gold diluent and then dilute it at 0.45 μl / mm 2 Add it to the above-mentioned glass fiber treated with mouse anti-human IgG polyclonal antibody, vacuum freeze-dry at -50°C for 5 hours, and store it at 4°C to make a marker pad.

[0063] ...

Embodiment 3

[0077] Example 3 HCV double-antigen sandwich method gold standard test strip with anti-human IgG monoclonal antibody added to sample pad or marker pad

[0078] Referring to Examples 1 and 2, the preparation of the sample pad or the marker pad was added with the HCV double-antigen sandwich method gold standard test strip of anti-human IgG monoclonal antibody, and finally obtained similar results with Examples 1.7 and 2.7, which shows that , Adding anti-human IgG monoclonal antibody or polyclonal antibody to the sample pad or marker pad can improve the specificity of gold-labeled test strip detection.

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Abstract

An improved double antigen sandwich immunoassay method aims to lower the false positive rate of the prior double antigen sandwich immunoassay method and improve the specificity of detection. The method can be applied to various detection methods such as rapid diagnosis, enzyme linked immune detection, chemiluminescent detection, and the like through adding an antihuman IgG monoclonal antibody or polyclonal antibody in a reaction system of the double antigen sandwich immunoassay method according to a certain proportion to participate in detecting. Compared with the prior double antigen sandwich immunoassay method, the double antigen sandwich immunoassay method of the invention remarkably lowers the detection background and the false positive rate and greatly improves the specificity.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to an improved double-antigen sandwich immunoassay. Background technique [0002] Immunoassay is a method of applying immunological techniques to determine the substances to be tested in specimens. In clinical testing, antibodies or antigenic substances in body fluids are mainly detected through antigen-antibody reactions. According to different detection principles, immunoassays can be divided into indirect method, sandwich method (including double antigen sandwich method and double antibody sandwich method), capture method, competition method and so on. [0003] The double-antigen sandwich method is a kind of sandwich method, which is widely used in the field of immunoassay. The basic principle is: use specific antigens to coat and prepare labeled conjugates, and form a coated antigen-antibody-labeled antigen complex through two immune combinations to complete the detection of the ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/558
Inventor 颜文豪潘少丽洪娟彭亮王宁燕王荣娥孙兴宝孙婧胡鹏
Owner GUANGDONG WESAIL BIOTECH CO LTD
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