Enzyme linked immunoreaction reagent kit for detecting rabies virus

Abstract

The disclosed enzyme linked immunity reagent for rabies virus comprises: the enzyme mark plate covered by rabies virus N protein, and the enzyme label as enzyme-labeled antibody. This invention has specificity up to 95%, sensitivity up to 0.25IU / ml, and precision (coefficient of variation C .V) up to 8.68%, and can complete the detection in one hour.

Description

technical field

[0001] The invention relates to an ELISA kit for detecting rabies virus. Background technique

[0002] Rabies is a zoonotic infectious disease of central nervous system infection caused by rabies virus, and the mortality rate after its onset is 100%. The death toll of rabies in my country ranks second in the world, second only to India. The case fatality rate and death toll of rabies rank first among Class A and Class B infectious diseases in my country.

[0003] According to the "National Rabies Surveillance Program", the domestic detection of rabies mainly adopts ① pathogen detection method: including immunofluorescence method for antigen detection and rapid rabies enzyme-linked immunosorbent assay for antigen detection; ② RT-PCR nucleic acid detection method; ③ virus isolation method: including Isolation of virus by cell culture method and inoculation of suckling mice; and ④ Antibody detection method: including specific antibody detection and neutralizin...

Examples

Embodiment 1

[0026] Embodiment 1, the preparation of the ELISA kit that detects rabies virus

[0027] 1. Preparation of enzyme-linked immunosorbent assay kit for detection of rabies virus

[0028] The ELISA kit of rabies virus consists of:

[0029] 1) A microtiter plate coated with rabies virus N protein

[0030] 2) Sample diluent: 10ml of a solution containing 2.1% NaCl and 4% PEG6000

[0031] 3) Positive serum 0.5ml

[0032] 4) Negative serum 0.5ml

[0033] 5) Horseradish peroxidase (HRP) labeled anti-dog IgG 10ml

[0034] 6) Concentrated washing liquid: 20ml

[0035] 7) Chromogenic agent: Chromogenic substrate A: 5ml of 3.3’-5.5’-tetramethylbenzidine solution; Chromogenic substrate B: 5ml of hydrogen peroxide solution;

[0036] 9) Stop solution: 5ml of 2mol / L sulfuric acid solution diluted with distilled water

[0037] The preparation method of above-mentioned each component is as follows:

[0038] 1. Preparation of an ELISA plate coated with rabies virus N protein

[0039] 1) ...

Embodiment 2

[0080] Embodiment 2, the effect experiment of the ELISA kit that detects rabies virus

[0081] 1. Kit specific detection:

[0082] Collect 20 rabies virus-negative sera and 20 rabies virus-positive sera, and use the fluorescent antibody virus neutralization test (FAVN) to determine the specific operation. Refer to the literature report (Cliquet F, Aubert M, Sagné L. Development of a fluorescent antibody virus neutralization test (FAVN) for the quantitation of rabies-neutralising antibody.Journal of ImmunologicalMethods, 1998,212:79-87) detected by the enzyme-linked immunoassay kit for the detection of rabies virus prepared by the method of Example 1, the results showed that in the positive serum Eighteen were positive, and 20 of the negative sera were negative, indicating a detection accuracy of 95%.

[0083] 2. Sensitivity detection: the standard rabies virus positive serum (Changchun Veterinary Research Institute of Military Medical Sciences) is gradiently diluted with the ...

technical field

[0001] The invention relates to an ELISA kit for detecting rabies virus. Background technique

[0002] Rabies is a zoonotic infectious disease of central nervous system infection caused by rabies virus, and the mortality rate after its onset is 100%. The death toll of rabies in my country ranks second in the world, second only to India. The case fatality rate and death toll of rabies rank first among Class A and Class B infectious diseases in my country.

[0003] According to the "National Rabies Surveillance Program", the domestic detection of rabies mainly adopts ① pathogen detection method: including immunofluorescence method for antigen detection and rapid rabies enzyme-linked immunosorbent assay for antigen detection; ② RT-PCR nucleic acid detection method; ③ virus isolation method: including Isolation of virus by cell culture method and inoculation of suckling mice; and ④ Antibody detection method: including specific antibody detection and neutralizin...

Examples

Embodiment 1

[0026] Embodiment 1, the preparation of the ELISA kit that detects rabies virus

[0027] 1. Preparation of enzyme-linked immunosorbent assay kit for detection of rabies virus

[0028] The ELISA kit of rabies virus consists of:

[0029] 1) A microtiter plate coated with rabies virus N protein

[0030] 2) Sample diluent: 10ml of a solution containing 2.1% NaCl and 4% PEG6000

[0031] 3) Positive serum 0.5ml

[0032] 4) Negative serum 0.5ml

[0033] 5) Horseradish peroxidase (HRP) labeled anti-dog IgG 10ml

[0034] 6) Concentrated washing liquid: 20ml

[0035] 7) Chromogenic agent: Chromogenic substrate A: 5ml of 3.3’-5.5’-tetramethylbenzidine solution; Chromogenic substrate B: 5ml of hydrogen peroxide solution;

[0036] 9) Stop solution: 5ml of 2mol / L sulfuric acid solution diluted with distilled water

[0037] The preparation method of above-mentioned each component is as follows:

[0038] 1. Preparation of an ELISA plate coated with rabies virus N protein

[0039] 1) ...

Embodiment 2

[0080] Embodiment 2, the effect experiment of the ELISA kit that detects rabies virus

[0081] 1. Kit specific detection:

[0082] Collect 20 rabies virus-negative sera and 20 rabies virus-positive sera, and use the fluorescent antibody virus neutralization test (FAVN) to determine the specific operation. Refer to the literature report (Cliquet F, Aubert M, Sagné L. Development of a fluorescent antibody virus neutralization test (FAVN) for the quantitation of rabies-neutralising antibody.Journal of ImmunologicalMethods, 1998,212:79-87) detected by the enzyme-linked immunoassay kit for the detection of rabies virus prepared by the method of Example 1, the results showed that in the positive serum Eighteen were positive, and 20 of the negative sera were negative, indicating a detection accuracy of 95%.

[0083] 2. Sensitivity detection: the standard rabies virus positive serum (Changchun Veterinary Research Institute of Military Medical Sciences) is gradiently diluted with the ...

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