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Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof

A technology for bronchitis and chicken infectivity, applied in the biological field, can solve the problems of unsuitability for vaccines, high cost, long use time, etc., and achieve the effects of easy industrial application, low cost, and simple operation.

Inactive Publication Date: 2012-10-10
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation is the most classic and reliable method for diagnosing this disease, but it takes a long time and is often missed due to the influence of other viruses infected at the same time; the agar diffusion test (AGP) is simple to operate, but its sensitivity is low, and the duration of precipitated antibodies is short. Not suitable for the evaluation of vaccine immune effect; virus neutralization test (VNT) can be used to detect IB antigens and antibodies, sensitive, specific, and can be used for virus typing, but the experimental operation is complicated, time-consuming and expensive; trace hemagglutination inhibition (HI) The experimental operation is simple, fast, easy to promote, and has the value of early diagnosis. However, chicken infectious bronchitis virus has no natural hemagglutination activity, and it must undergo special treatment to agglutinate red blood cells, which also limits the use of HI in the detection of IBV antibodies. The application of RT-PCR detection method has the characteristics of sensitivity, rapidity and specificity, but it has high requirements on laboratory conditions, complicated operation, and is difficult to popularize

Method used

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  • Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof
  • Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof
  • Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation, purification and properties of monoclonal antibody against nephrophilic avian infectious bronchitis virus DS10 strain (referred to as nephrophilic IBV DS10 strain)

[0052] 1. Nephrophilic IBV DS10 strain

[0053] The preservation information of the nephrophilic IBV DS10 strain used in the present invention is as follows:

[0054] Biological material (virus strain): Ck / Jiangsu / DS10 / 2008

[0055] Taxonomic designation: chicken infectious bronchitis virus;

[0056] Latin name: Infectious Bronchitis Virus (IBV);

[0057] Preservation unit: General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee;

[0058] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing;

[0059] Date of deposit: June 25, 2010;

[0060] Deposit number: CGMCC No. 3957.

[0061] Note: In this article, for the convenience of expression, the strain with ...

Embodiment 2

[0086] Example 2 Preparation of standard IBV antibody positive serum and negative serum

[0087] The invention prepares standard IBV antibody positive serum and negative serum, which are used for the optimization of detection conditions and the judgment of detection results.

[0088] (1) Standard IBV antibody positive serum: preparation of nephrophilic IBV DS10 strain inactivated oil emulsion vaccine (Veterinary Drug Evaluation Center Group of the Ministry of Agriculture, etc. Compilation of Quality Standards for Veterinary Biological Products (2010) [M]. Beijing: China Agricultural Press , 2011: 69-73), immunized SPF chickens (purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd. eggs, hatched and raised in our laboratory), and prepared standard IBV antibody-positive serum. The specific method is as follows: SPF chickens are raised in an isolated environment until they are 14 days old, and immunized with nephrophilic IBV DS10 strain inactivated oil emu...

Embodiment 3

[0090] Example 3 Establishment and standardization of an indirect ELISA method for detecting nephrophilic IBV antibodies (or antigens)

[0091] In order to establish an indirect ELISA kit for detecting nephrophilic IBV antibody (or antigen), the following conditions were optimized for the indirect ELISA detection method for nephrophilic IBV antibody (or antigen):

[0092] (1) Square array experiment to determine the optimal coating concentration of anti-nephrophilic IBV DS10 monoclonal antibody and the optimal dilution of serum

[0093] Anti-nephrophilic IBV DS10 monoclonal antibody was determined by square array titration method (Qiu Dexin et al. Indirect ELISA for detection of porcine pseudorabies serum antibody [J]. Chinese Journal of Veterinary Medicine, 2002, 22 (3): 149-151), and the standard was positive , The best working concentration of negative serum. Take a 96-well ELISA plate, and dilute the anti-nephrophilic IBV DS10 strain monoclonal antibody solution (prepared...

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Abstract

The invention provides an indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting a nephropathogenic avian infectious bronchitis virus and an antibody thereof, and belongs to the technical field of biology. The indirect ELISA kit for detecting the nephropathogenic avian infectious bronchitis virus and the antibody thereof comprises an ELISA plate which is coated by DS10 monoclonal antibodies resisting the nephropathogenic avian infectious bronchitis virus and is combined with DS10 inactivated purified antigens resisting the nephropathogenic avian infectious bronchitis virus. The indirect kit for detecting the nephropathogenic avian infectious bronchitis virus and the antibody thereof has the advantages of specificity, sensitivity, quickness and simplicity and convenience in operation, and can be used for quickly diagnosing whether chickens are infected by the nephropathogenic avian infectious bronchitis virus or not and monitoring the immune antibody in actual production.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to antibody engineering technology, in particular to an indirect ELISA kit for detecting nephrophilic chicken infectious bronchitis virus and its antibody. Background technique [0002] Chicken Infectious Bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious infectious disease of chickens caused by Infectious Bronchitis Virus (IBV) of the genus Coronavirus, which can cause chicken respiratory tract infection, Nephritis and decreased egg production are the main features, which can cause the mortality rate of young chickens as high as 40%, and the egg production of adult chickens will decrease by 10%-50% after infection, which will bring significant economic losses to the world's chicken industry. [0003] Chicken infectious bronchitis virus has a variety of serotypes and multi-tissue tropism. Since the first report of respiratory type IB in 1931, subsequent reports of ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
Inventor 陆吉虎高峰唐应华杨维维侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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