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Rabies virus antibody test paper, preparation method thereof and detection method thereof

A rabies virus and antibody detection technology, applied in the field of rabies virus antibody detection test strips and their preparation, can solve problems such as false positives, and achieve the effects of avoiding false positives, practical and effective detection methods, and improving accuracy and sensitivity

Active Publication Date: 2019-07-02
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The marker antigen used in the current rabies virus antibody detection test strip is the whole virus of rabies virus, and the rabies vaccine is also a whole virus vaccine. Since it is impossible to achieve 100% purification, there will be virus fragments, bovine serum and cell components in the vaccine. , the antibodies produced in vivo include antibodies against 5 structural proteins (M, G, N, P, L) of rabies virus, antibodies against bovine serum antibodies and antibodies against cell components, and when the whole virus is used as the labeled antigen, it cannot Avoid the presence of residual components such as the above-mentioned virus fragments, bovine serum, and cell fragments in the labeled antigen. Therefore, the use of the whole virus-labeled rabies virus test strip to bind to the blood sample is not necessarily neutralizing antibodies, and there is a risk of false positives

Method used

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  • Rabies virus antibody test paper, preparation method thereof and detection method thereof
  • Rabies virus antibody test paper, preparation method thereof and detection method thereof
  • Rabies virus antibody test paper, preparation method thereof and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation and identification of rabies virus-like particles

[0037] (1) Material

[0038] The donor plasmid pFastBac1 was purchased from Invitrogen Company, and the strain E.coli DH10Bac, insect cell Sf9, and rabies virus were provided by Changchun Zhuoyi Biological Co., Ltd.

[0039] Rabbit anti-RABV MP serum was made by Changchun Sino Biotechnology Co., Ltd.; Phusion ultra-fidelity DNA polymerase and T4 DNA ligase were purchased from NEB Company; restriction endonuclease and protein marker were purchased from Thermo Company; DH5α competent cells , DNA Molecular Marker, and BacPAK Baculovirus Rapid Titer Detection Kit were purchased from Takara Company; plasmid mini-extraction kits, DNA gel recovery kits, and genome extraction kits were purchased from Axygen Company; cell transfection reagent CellfectionII Reagent was purchased from Invitrogen Company; fetal bovine serum is a product of GIBCO Company.

[0040] (2) Method

[0041] (a) Extraction of viral...

Embodiment 2

[0064] Preparation and identification of embodiment 2 rabies virus G protein monoclonal antibody

[0065] (1) Select 5 female BALB / c mice aged 6-8 weeks, inject the aforementioned purified rabies virus G protein virus-like particles and the same amount of Freund's complete adjuvant emulsion into the mice, and inject 200 μl subcutaneously into each mouse . Two weeks later, the second immunization was carried out, and the mice were injected with rabies virus G protein virus-like particles and an equal amount of Freund's incomplete adjuvant emulsion, and the injection volume and method were the same as the first immunization. The third immunization was carried out two weeks after the second immunization, and the immunization method and virus injection volume were the same as the second immunization. Two weeks after the third immunization, when the serum ELISA titer reached 1:10000 or more after the tail-cut blood collection, the splenocytes and myeloma cells were removed at 6×10...

Embodiment 3

[0067] The preparation of embodiment 3 rabies virus antibody quantitative detection kit

[0068] (1) Preparation of sample pad

[0069] The purified rabies virus-like particles prepared in Example 1 were diluted to 1.0 mg / ml with 20 mM sodium tetraborate solution, containing 2% Tween-20, and sprayed on the glass cellulose membrane as a sample pad.

[0070] (2) Preparation of gold standard pad

[0071] With 0.2mol / L K 2 CO 3 After adjusting the pH value of the 25nm colloidal gold solution to 8.4, add the rabies virus-like particle monoclonal antibody to the colloidal gold solution under rapid stirring according to the protein concentration of 50 μg / ml of the qualified rabies virus-like particle monoclonal antibody after purification And continue to stir for 30 minutes; add 10% BSA to a final concentration of 1%, stir for 30 minutes, centrifuge at 10,000r / min for 30 minutes, carefully suck off the supernatant, and the precipitate is the initially purified gold-labeled rabies ...

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Abstract

The invention discloses rabies virus antibody test paper and a preparation method thereof and a detection method thereof, which belong to the technical field of antibody test paper. The problems of false positive risk and safety hazard in the prior art are solved. The test paper comprises, from one end to the other end, a sample pad which coats a rabies virus antigen, a gold standard pad which coats a monoclonal antibody against the rabies virus antigen, a nitrocellulose membrane which coats a detection line and a quality control line, and an absorbent pad, wherein the sample pad, the gold standard pad, the nitrocellulose membrane and the absorbent pad overlap each other and are attached to the upper surface of a backboard. The rabies virus antigen coated by the sample pad is rabies virus-like particles. According to the invention, the used labeled antigen is the rabies virus-like particles expressed by insect cells, which avoids false positives; the monoclonal antibody against the rabies virus G protein competes with a neutralizing antibody in the blood to improve the detection accuracy and sensitivity; the virus-like particles are free of nucleic acid components; and biosafety risks produced by the use of whole viruses as marker antigens are eliminated.

Description

technical field [0001] The invention relates to the technical field of antibody detection test paper, in particular to a rabies virus antibody detection test paper and a preparation method and a detection method thereof. Background technique [0002] At present, the commonly used antibody detection methods in the world include enzyme-linked immunosorbent assay, rapid fluorescence focus inhibition test, neutralization immunofluorescence test, mouse neutralization test, etc., all of which require a high test environment, sophisticated test instruments, and professional expertise. Operators and other conditions make it impossible to monitor the rabies virus antibody level conveniently and quickly, which is not conducive to large-scale implementation. [0003] Immune colloidal gold technique (Immune colloidal gold technique) is a new type of immune labeling technology that uses colloidal gold as a tracer marker to apply to antigens and antibodies. It is convenient and fast to us...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/58G01N33/531
CPCG01N33/56983G01N33/583G01N33/531Y02A50/30
Inventor 殷玉和石晶刘伟王慧慧王倩丁秋雨赵玉环倪婷婷王莹夏振强
Owner CHANGCHUN SR BIOLOGICAL TECH
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