ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof

A kit and rabies technology, applied in the field of molecular biology and biology, can solve the problems of high detection cost, unfavorable promotion and use, maintenance of biological macromolecular activity, and low antigen purity, so as to avoid false negative results, low cost, and high sensitivity sexual effect

Inactive Publication Date: 2012-06-27
TANGSHAN YIAN BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA detection method has higher requirements on the purity of the coated antigen. The methods for purifying rabies virus in the prior art mainly include ion exchange chromatography purification, gel (molecular sieve) chromatography purification, etc. The disadvantage is that the purity of the antigen is relatively high. low cost and complex operation, which is not conducive to maintaining the activity of biological macromolecules and saving costs. Therefor

Method used

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  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 kit

[0030] The preparation and coating of coated antigens adopts microcarrier suspension culture technology to culture Vero cells. After the cells grow into a single layer, they are inoculated with rabies virus CTN-1 strain virus according to a certain ratio, purchased from China Institute for the Control of Pharmaceutical and Biological Products, and cultured. and harvest virus fluid. The linear sucrose gradient zonal centrifugation, concentration, extraction and purification of virus fluid includes the following steps:

[0031] 1) After Vero cells grow into a monolayer on the microcarrier, inoculate rabies virus at a ratio of 0.03 multiplicity of infection (M.O.I), at a temperature of 33°C, a rotating speed of 40r / min, pH 7.6, dissolved oxygen 50%, and adding 0.05% bovine serum white The 199 medium of the protein is cultured under medium conditions, and the supernatant is harvested after 4-6 days of culture until the cells are comple...

Embodiment 2

[0047] The assay procedure of rabies neutralizing antibody titer in embodiment 2 serum

[0048] Reconstitute positive serum and negative serum with 0.5ml sample diluent, and serially dilute to 1:100 by 10 times.

[0049] Reconstitute the standard serum with 0.5ml of sample diluent, obtain the standard curve according to the following dilution method, first dilute the standard serum to 0.4IU / ml, then dilute to 0.04IU / ml by 1:10, and perform 2 times on this basis Dilute to obtain the following gradient standard serum: 0.04IU / ml, 0.02IU / ml, 0.01IU / ml, 0.005IU / ml, 0.0025IU / ml, 0.00125IU / ml, 0.000625IU / ml.

[0050] Serum to be tested was serially diluted 10 times to 1:100.

[0051] Add diluted standard sera, negative sera, positive sera and samples to be tested with different gradient titers to the wells of the plate, 100ul / well, repeat in 2 wells. Incubate in a constant temperature incubator at 37°C for 30 minutes. Dilute the concentrated washing solution 20 times with deionize...

Embodiment 3

[0055] Embodiment 3 The formulation and characteristic evaluation of kit quality standard of the present invention

[0056] 1. Sensitivity: Dilute the OIE rabies positive serum (6.7IU / ml) with the sample diluent to 0.4IU / ml, then dilute it 10 times to 0.04IU / ml, then dilute it by 2 times, take 0.04, 0.02, 0.01, 0.005, 0.0025, 0.00125, 0.000625, 0.0003125, 0.00015625IU / ml titer serum, set up a negative control at the same time, repeat 2 wells for each dilution, follow the kit operation steps, get higher than the negative control serum OD value corresponding The serum antibody titer of the kit is the minimum detection limit of the rabies virus antibody of the kit, and the results are shown in Table 1.

[0057] Table 1 Test results of OIE rabies positive serum kit after doubling dilution

[0058]

[0059] Note: PC is positive control serum, NC is negative control serum.

[0060] It can be seen from the above table that when the OIE rabies positive serum is 0.015625IU / ml, the...

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Abstract

The invention discloses an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of an animal rabies neutralizing antibody. Rabies virus totivirus particles purified by using a linear sucrose gradient zonal centrifugation method are coated on an enzyme label plate of the kit. The coating has high antigen purity and is capable of detecting antibodies generated by proteins, such as rabies virus glycoprote, nucleoprotein and the like. Found from researches, the kit disclosed by the invention has specificity of being up to 100%, minimum detectable quantity of sensitivity of 0.0625 IU/ml and a variation coefficient CV (repeatability) of 2.74%. The kit has good stability, simple operation and low cost and is applied to popularization and application.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a kit for detecting animal rabies neutralizing antibodies by an indirect enzyme-linked immunosorbent assay (ELISA) and a preparation method thereof. Background technique [0002] Rabies is a zoonotic acute and highly fatal infectious disease caused by rabies virus (RV) infection, which is widely prevalent all over the world. According to the latest incomplete statistics of the World Health Organization (WHO), approximately 55,000 people died of rabies, and the number of deaths in my country ranks second in the world every year, and the number of deaths has remained high in recent years. It is the spread of rabies virus among animals that directly leads to the prevalence of human rabies. To prevent human rabies, the first thing to do is to control and eliminate animal rabies, that is, to eliminate the source of infection. Therefore, in order to fundamentally pre...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531
Inventor 王海霞陈静岳雷李淑芬张振山
Owner TANGSHAN YIAN BIOLOGICAL ENG CO LTD
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