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Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting duck flavivirus serum antibody

A technique for duck flavivirus disease and serum antibody, which is applied in the field of zooinfectious diseases, can solve the problems of rough method, unsuitable clinical application and promotion, unclear data, etc., and achieves the effect of a simple detection method.

Inactive Publication Date: 2012-03-21
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, Su [4] etc. have initially established an ELISA method for detecting antibodies against duck yellow virus disease, but the established method is relatively rough, the data is unknown, and various conditions have not been optimized, so it is not suitable for clinical application and promotion

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Duck yellow virus antigen of the present invention is obtained in the following manner:

[0016] (1) Concentration of antigen

[0017] A flavivirus WR strain with good immune activity isolated and identified by our laboratory with semi-species embryos (the preservation number of the strain is CGMCC No.4906, derived from an invention patent applied by the applicant: the application number is 201110143407.6, a duck breeding duck yellow virus and its inactivated vaccine), the embryo allantoic fluid containing the virus was collected and centrifuged at 4°C, 8000 r / min for 30 min, the supernatant was centrifuged at 45000 r / min for 2 h at 4°C, and the supernatant was discarded The pellet was resuspended with sterilized PBS to obtain a virus concentrate, which was stored at -70°C.

[0018] (2) Antigen purification

[0019] Antigens are purified by concentration through sucrose gradient centrifugation and ultracentrifugation. Five gradient sucrose balance solutions of 10%, 2...

Embodiment 2

[0021] Duck yellow virus disease positive serum and negative serum of the present invention are obtained in the following manner:

[0022] (1) Positive serum

[0023] The WR strain virus purification solution (the virus purification solution obtained in step (2) of Example 1) was inactivated with formaldehyde, emulsified with Freund's complete adjuvant at a ratio of 1:1, and then immunized adult laying ducks with an interval of 15 days After the third immunization, blood was collected from the heart, and the serum was collected by centrifugation. After inactivation at 56°C, it was proved to be duck yellow virus disease positive serum by neutralization test, and it was ready for use.

[0024] (2) negative serum

[0025] The whole blood of healthy ducks not infected with duck yellow virus or immunized with duck yellow virus disease vaccine was collected, the supernatant was collected after centrifugation, inactivated at 56°C, and proved to be duck yellow virus disease negative ...

Embodiment 3

[0027] The optimal antigen antibody concentration, enzyme-labeled secondary antibody concentration and critical value of duck yellow virus disease antibody indirect ELISA detection method of the present invention are obtained by the following methods:

[0028] (1) Optimal concentration of antigen and antibody

[0029] Determined by the matrix method. Use the purified antigen (purified virus) with coating solution at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600 Diluted, coated on 96-well Costar microtiter plate, after overnight at 4°C, washed 3 times with PBST, blocked with 5% skimmed milk at 37°C for 2 h, washed 3 times with PBST, diluted 20 times with PBS, 40 times, 80 times, 160 times, 320 times and 640 times, each negative and positive serum titer was repeated 4 times, reacted with the coated antigen for 2 hours, washed 3 times with PBST, and reacted with 1000 times enzyme-labeled secondary antibody After 1 h, wash 3 times, develop color with TMB f...

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Abstract

The invention relates to an indirect enzyme-linked immunosorbent assay (ELISA) method for detecting a duck flavivirus serum antibody, which is characterized in that: duck flavivirus is used as an envelop antigen, and an indirect ELISA method for detecting duck flavivirus serum antibody in duck blood serum is established. The method can effectively diagnose the duck flavivirus which is prevalent at present in China and can carry out large-scale investigation of blood serum epidemiology, and a quick, simple and convenient detection method can be provided for controlling the prevention and the prevalent situation of the duck flavivirus.

Description

technical field [0001] The invention relates to an indirect ELISA method for detecting serum antibodies against duck yellow virus disease, belonging to the field of animal infectious diseases. Background technique [0002] Since April 2010, a new disease caused by a flavivirus that caused a sudden drop in laying duck eggs has occurred successively in Jiangsu, Zhejiang, Shandong, Henan, Fujian and other places in my country, which can cause laying ducks to eat A rapid decline in production, a rapid decline in egg production rate, and even production stoppage and death in varying degrees. Ducks with a high egg production rate will drop rapidly from 80% to 95% of the egg production peak to 20% to 30% about 4 to 5 days after the disease, and in severe cases, production may stop around 7 days after the onset; (Egg) The egg production rate of ducks increases slowly or decreases, and the egg production rate is low for a long time or there is no egg production peak. The incidence r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
Inventor 施少华黄瑜万春和傅光华程龙飞陈红梅梁昭平王斌李敏王鑫许芬芬
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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