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Application of WHsAg monoclonal antibody as ELISA detection reagent

A monoclonal antibody and detection reagent technology, applied in the biological field, can solve problems such as self-exploration and preparation

Pending Publication Date: 2021-10-29
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of HBsAg has been commercialized, including ELISA kits for humans, rats, mice, pigs, guinea pigs, etc., but there is no commercial WHsAg ELISA kit, and the detection methods and required reagents can only be explored and prepared by ourselves , the patent of this invention has important application value for the preparation of WHsAg ELISA kit

Method used

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  • Application of WHsAg monoclonal antibody as ELISA detection reagent
  • Application of WHsAg monoclonal antibody as ELISA detection reagent
  • Application of WHsAg monoclonal antibody as ELISA detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] a. Select a known polyclonal antibody-anti-WHsAb (7.2mg / ml), dilute it with Coating Buffer 1:720, 100μl / well, coat the ELISA plate, and overnight at 4°C;

[0042] b. Take out the coated ELISA plate the next day, and wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0043] c. Blocking Buffer, 300 μl, 37°C, 1 hour;

[0044] d. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0045] e. Add known WHsAg positive serum samples and negative controls, serum samples were diluted 1:10 with Dilution Buffer, 100 μl, 37°C, 1 hour;

[0046] f. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0047] g. Add HRP-labeled monoclonal antibody T9G10D4, original concentration 1mg / ml, and T1C3C9 (source of HRP labeling method: HRP Conjugation Kit, 701-0030), diluted with Dilution Buffer, the dilution ratios are 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 50μl, 37°C, 45 minutes;

[0048] h. Washing Buffer 4 times, 3 minutes each time; ...

Embodiment 2

[0054] a. Add monoclonal antibody T1C3C9 as coating antibody, dilute with Coating Buffer 1:1000, 1:2000, 1:4000, 1:10000 respectively, 100 μl / well, coat ELISA plate, 4 ℃ overnight;

[0055]b. Take out the coated ELISA plate the next day, and wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0056] c.Blocking Buffer closed, 37 ℃, 1 hour;

[0057] d. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0058] e. Add known WHsAg positive serum samples and negative controls, serum samples were diluted 1:10 with Dilution Buffer, 100 μl per well, 37°C, 1 hour;

[0059] f. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0060] g. Add HRP-labeled monoclonal antibody T9G10D4, the original concentration is 1mg / ml, (source of HRP labeling method: HRP Conjugation Kit, 701-0030), diluted with Dilution Buffer, the dilution ratio is 1:20000, 50μl, 37℃, 45 minutes;

[0061] h. Washing Buffer 4 times, 3 minutes each time;

[0062] i. Add ...

Embodiment 3

[0067] The specific method of WHsAg ELISA before improvement:

[0068] a. Use a known polyclonal antibody, anti-WHsAb (7.2mg / ml), dilute it with Coating Buffer 1:720, 100μl / well, coat the ELISA plate, and leave overnight at 4°C;

[0069] b. Take out the coated ELISA plate the next day, and wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0070] c.Blocking Buffer closed, 37 ℃, 1 hour;

[0071] d. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0072] e. Add the serum sample to be tested and the negative and positive controls, dilute the serum sample with Dillution Buffer 1:10, 100 μl per well, 37 degrees, 1 hour;

[0073] f. Wash the plate with Washing Buffer 4 times, 3 minutes each time;

[0074] g. Add Biotin-labeled anti-WHsAb (7.2mg / ml), dilute with Dillution Buffer 1:300, 37 degrees, 45 minutes;

[0075] h. Washing Buffer 4 times, 3 minutes each time;

[0076] i. Add HRP-Avidin, dilute with Dilution Buffer 1:200, 37°C, 45 minutes; ...

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Abstract

The invention relates to application of an anti-WHsAg monoclonal antibody as an ELISA (enzyme-linked immunosorbent assay) detection reagent, in particular to preparation of the anti-WHsAg monoclonal antibody which is suitable for WHsAg ELISA detection and comprises an enzyme-labeled antibody T9G10D4 and a coated antibody T1C3C9, and the optimal use concentration of the enzyme-labeled antibody T9G10D4 and the coated antibody T1C3C9 when the enzyme-labeled antibody T9G10D4 and the coated antibody T1C3C9 are applied to WHsAg ELISA is determined. Compared with a detection method before improvement, namely a detection method using an anti-WHsAg polyclonal antibody and adopting a biotin-avidin system, the improved method not only can optimize the detection process and shorten the detection time, but also can remarkably improve the OD value of a positive sample, reduce the OD value of a negative sample and improve the detection effect, the invention has an important application value in preparation of a WHsAg ELISA kit, and a basic research tool of a WHV infected marmot model is perfected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to two kinds of monoclonal antibodies that can be used in WHsAg ELISA detection and the optimal concentrations of coated antibodies and enzyme-labeled antibodies. Background technique [0002] At present, there are still many problems to be solved in the treatment of chronic hepatitis B, and its related research is inseparable from suitable animal models. Woodchuck hepatitis virus (WHV) is similar to hepatitis B virus (HBV) in terms of virus morphology, genome structure, and replication cycle. WHV can naturally infect marmots, and the natural course of infection is similar to that of HBV infection is similar, so marmot is an ideal animal model for studying HBV. [0003] Hepadnavirus surface antigen (HBsAg, WHsAg) is the coat protein of hepadnavirus, and is a sign of current infection of hepadnavirus. At present, the detection of HBsAg has been commercialized, including ELIS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576G01N33/577G01N33/535
CPCG01N33/5764G01N33/577G01N33/535G01N2333/02
Inventor 王宝菊王冬初杨东亮李蒙蒙李金杨雪晟冯雪梅李潇冉邓辉
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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