Porcine reproductive and respiratory syndrome virus (PRRSV) double-antibody sandwich ELISA kit

A respiratory syndrome and double-antibody sandwich technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of many factors affecting the test results, unfavorable promotion and use at the grassroots level, and achieve the goal of being suitable for large-scale promotion and application and low cost of use , good repeatability

Inactive Publication Date: 2010-03-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These three technology patents all involve molecular biology technology, and there are many factors affecting the test results, and because of the cost factor, it is not conducive to the promotion and use at the grassroots level

Method used

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  • Porcine reproductive and respiratory syndrome virus (PRRSV) double-antibody sandwich ELISA kit
  • Porcine reproductive and respiratory syndrome virus (PRRSV) double-antibody sandwich ELISA kit
  • Porcine reproductive and respiratory syndrome virus (PRRSV) double-antibody sandwich ELISA kit

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Experimental program
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Embodiment 1

[0028] The establishment of embodiment 1 double antibody sandwich ELISA method

[0029] 1. Preparation and preliminary screening of monoclonal antibodies

[0030] experimental method

[0031] 1 Preparation of monoclonal antibodies

[0032] MARC-145 cells were cultured for cytotoxicity according to the conventional method. After 90% lesions (CPE) were produced, the cells were frozen and thawed three times. The virus liquid was collected, frozen and thawed three times, centrifuged at 12000rpm / min for 30min, and the supernatant was discarded. After ultracentrifugation, harvest the precipitate as crude virus, dissolve the precipitate with PBS, and measure the protein content of the virus with a spectrophotometer, according to the formula protein concentration (mg / ml)=1.45×OD 280nm -0.74×OD 260nm , to calculate the protein concentration of the crude virus extracted by ultracentrifugation. BALB / c mice were immunized with crude virus as antigen.

[0033] The PRRSV BJ-4 cytotoxic...

Embodiment 2

[0208] The composition of embodiment 2 test kits

[0209] The composition of the kit in this example is as follows:

[0210] ELISA plate: coated with monoclonal antibody secreted by N36 strain

[0211] Enzyme-labeled antibody: horseradish peroxidase-labeled monoclonal antibody produced by N35 secretion

[0212] Lysis solution: 1% Triton X-100, 2.8M Tris-base, 800mM NaCl, pH7.5.

[0213] Substrate solution: 100mmol / L citric acid solution (21g citric acid C 6 h 6 o 7 ·H 2 O was dissolved in deionized water, and the volume was adjusted to 1L) 24.3mL, 200mmol / L Na 2 HPO 4 12H 2 O(71.6gNa 2 HPO 4 .12H 2 O (dissolved in deionized water, dilute to 1L) 25.7mL and mix well, add 50mg of tetramethylbenzidine (TMB), add 50μL of 30% H 2 o 2 ;

[0214] Stop solution: (2mol / L H 2 SO 4 ) respectively take 177.8mL of distilled water and 22.2mL of concentrated sulfuric acid and mix them.

[0215] Add 0.5mL Tween-20 to the washing solution 1000mL 10mmol / L PBS with pH 7.4;

[0216...

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Abstract

The invention provides a porcine reproductive and respiratory syndrome virus double-antibody sandwich ELISA kit. The kit comprises: an elisa plate coated with PRRSV N protein monoclonal antibody, an enzyme labeling PRRSV N protein monoclonal antibody, lysis solution and the like. A capture antibody and a detection antibody are respectively aimed at antigenic determinants with different N proteins.The kit provides a reliable means for quick detection of clinical PRRSV antigen. The kit can detects that blood serum only contains 0.2 TCID50 highly pathogenic PRRSV JXwn06 strain (non-highly pathogenic strain can be also be detected). Through detecting clinically collected 80 blood serum samples, compared with RT-PCR result, the specificity of the method is 88 percent, the sensitiveness is 90 percent and the coincidence rate of the specificity and the sensitiveness are 88.8 percent. The kit is convenient for operation, low in use cost, good repetitiveness and suitable for wide promotion andapplication.

Description

technical field [0001] The invention relates to an ELISA detection kit, in particular to a double-antibody sandwich ELISA kit for detecting porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) mainly causes reproductive disorders such as abortion, premature birth and stillbirth in pregnant sows, and respiratory symptoms in piglets and fattening pigs. It is a highly contagious infectious disease (Albina, 1997 ; Christianson et al., 1992; Mengeling et al., 1998; Rossow, 1998). The disease first broke out in the U.S. in 1987 (Keffaber K K., 1989). In just over ten years, it spread rapidly to all countries and regions with developed pig farming in the world, bringing huge economic benefits to the pig farming industry in the world. loss. The occurrence of the disease was first reported in my country in 1996, and it was proved to be cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 杨汉春郭鑫刘从敏盖新娜陈艳红查振林
Owner CHINA AGRI UNIV
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