Rabies virus diagnostic reagent kit

A diagnostic kit, rabies virus technology, applied in biological testing, material testing products, measuring devices, etc., can solve the problems of decreased detection sensitivity and difficult purification of the whole virus.

Inactive Publication Date: 2012-10-10
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole virus has the disadvantage of not being easy to purify, which will lead to a decrease in detection sensitivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A rabies virus diagnostic kit, comprising:

[0022] 1) ELISA plate;

[0023] 2) Coating solution: pH 9.4, 50 mmol / L carbonate buffer (CB);

[0024] 3) Enzyme marker: horseradish peroxidase-labeled mouse anti-rabbit enzyme-labeled antibody;

[0025] 4) Chromogenic agent: TMB (3.3.5.5 tetramethylbenzidine) chromogenic solution;

[0026] 5) Washing solution: pH 7.2 PBST phosphate buffer solution;

[0027] 6) Substrate solution: PH5.0 jujube citric acid phosphate

[0028] 7) Stop solution: 2M H 2 SO 4 ;

[0029] 8) Negative control: negative serum of rabies virus standard dog;

[0030] 9) Positive control: Positive serum of rabies virus standard dog.

Embodiment 2

[0032] A kind of rabies virus diagnostic kit, the process of this diagnostic kit working optimal condition optimization is as follows:

[0033] Judgment basis for the best reaction conditions: process the data of each reaction, calculate the P / N value of the reaction, take the largest P / N value, and the P value close to 1 as the judgment basis for the best reaction conditions of indirect ELISA P / N value Calculation method: P / N = (average OD450 nm value of positive control wells - average OD450 nm value of blank control wells) / (average OD450 nm value of negative control wells - average OD450 nm value of blank control wells), characterized by: the ultraviolet light wave is 450nm.

[0034] 1. Selection of the best antigen coating solution

[0035] 50 mmol / L pH 9.4 carbonate buffer (CB), 20 mmol / L pH 8.5 Tris-Hcl (TB), pH 7.2 PBS buffer, pH 7.2 PBST buffer, distilled water (W), normal saline ( S) As a coating solution, dilute the rabies virus protein at a fixed concentration (8...

Embodiment 3

[0048] Embodiment 3: the effect experiment of rabies virus diagnostic kit

[0049] 1. Determination of critical value

[0050]40 samples of normal chicken serum (SPF) were randomly selected, tested according to the above-mentioned indirect ELISA method, and the average OD value (x) and standard deviation SD of the 40 samples of serum were calculated. + 3SD, it can be judged as positive at the 99.9% level. According to statistics, the average value of 40 negative sera is 0.210, and the standard deviation is 0.024, then the critical value = 0.210+3×0.024=0.282. It can be seen that when the OD450 value of the sample is ≥0.281, it can be tested with 99% confidence When the OD450 value of the sample is less than 0.281, it can be judged as negative with 99% confidence.

[0051] 2. Specificity test

[0052] The canine distemper, canine parvovirus, and canine parainfluenza virus positive sera were tested according to the above ELISA operating procedures, and the rabies virus positi...

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Abstract

The invention provides a rabies virus diagnostic reagent kit. The working conditions of the diagnostic reagent kit are optimized into the following optimal conditions that the optimal coating solution is 50mmol/L carbonate buffer solution with the pH value of 9.4; the optimal confining liquid concentration is 1 percent of BSA-PBS (Bovine Serum Albumin-Phosphate Buffer Solution); the optimal antigen coating quantity is 8g/mL; the optimal dilution of a serum is 1:300; the optimal action time of the serum is 1h; the optimal dilution of a secondary antibody is 1:5,000; the optimal action time of the secondary antibody is 1h; and the optimal color development of a substrate is 25min. According to the reagent kit, an envelope protein expressed by a rabies virus gene replaces a totivirus coated elisa plate so as to avoid the virus dispersing risk of the antibody, which is generated due to the case that a great amount of purified viruses are cultured for coating. The reagent kit has high sensitivity, high accuracy and low production cost, and is simple, rapid and convenient to operate.

Description

technical field [0001] The invention relates to a rabies virus diagnostic kit, which belongs to the technical field of veterinary biological diagnosis. Background technique [0002] Rabies is a zoonotic disease caused by the rabies virus. The case fatality rate is extremely high. Once the disease occurs, almost all of them die, causing symptoms such as encephalomyelitis, also known as hydrophobia. [0003] At present, there are certain researches and reports on the methods and corresponding kits for rabies virus antibody detection, and certain products are also listed. However, existing methods for detecting rabies virus antibodies, including common ELISA methods, fluorescent immunoassays, cell culture methods and PCR techniques, have corresponding weaknesses. Ordinary ELISA kits mostly use prokaryotic expressed recombinant proteins or cultured whole viruses as coating antigens. Whole virus has the disadvantage that it is not easy to purify, which will lead to a decrease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
Inventor 吴红云徐荣君韩改会
Owner ZHENGZHOU HOUYI PHARMA
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