Improved double-antigen sandwiching immunity detection method

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

Active Publication Date: 2009-08-05
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-labeled 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 NS3 part of the hepatitis C virus gene as the DNA segment corresponding to SEQ ID NO.1 (part of the NS3 protein), and its upstream primer is as shown in SEQ ID NO.2 with a BamHI site, and downstream primers As shown in SEQ ID NO. 3, with HindIII site. After the PCR fragment was recovered, it was digested with BamHI and HindIII, and then ligated into the PQE30 vector (Qiagen) 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 cultured with 500ml LB medium containing 100ug / ml ampicillin sodium (Shanghai Shenggong Bioengineering Technology Service Co., Ltd., catalog number A0339) at 37°C with shaking to OD600= About 1.0, IPTG (Shanghai Shenggong Biol...

Embodiment 2

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

[0059] Refer to Examples 1.1, 1.2, 1.3, and 1.4 to prepare HCV coated 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 with PBS buffer to 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0 mg / ml, and soak glass fibers (Watman), -50 Store at 4℃ after freeze-drying in vacuum for 6h. PBS without mouse anti-human IgG polyclonal antibody was used as a control.

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

[0063] Dilute the HCV coating antigen PQE-NS3 and the contro...

Embodiment 3

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

[0078] Refer to Examples 1 and 2 to prepare HCV double-antigen sandwich gold-labeled test strips with sample pads or marker pads with anti-human IgG monoclonal antibodies. Finally, similar results to Examples 1.7 and 2.7 were obtained, 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 immunoassays, in particular to an improved double-antigen sandwich immunoassay method. Background technique [0002] Immunoassay is a method of applying immunological techniques to determine the substance to be tested in a specimen. In clinical tests, antibodies or antigenic substances in body fluids are mainly detected by antigen-antibody reaction. According to different detection principles, immunoassay 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: coating with a specific antigen and preparing a labeled conjugate, and forming a coated antigen-antibody-labeled antigen complex through two immunizations to complete the detection of the antibody ...

Claims

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Application Information

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Patent Type & Authority Applications(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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