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ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit

A technology for respiratory syndrome and detection kit, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problem of rarely detecting PRRSVIgM antibodies, and achieve the effect of improving sensitivity and specificity, strong specificity, and overcoming low sensitivity

Inactive Publication Date: 2013-10-23
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can detect PRRSV or antibody levels, they rarely detect PRRSV IgM antibodies

Method used

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  • ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit
  • ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit
  • ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as preparation method and application of ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of ELISA Kit for Detection of Porcine Reproductive and Respiratory Syndrome Virus IgM Antibody

[0053] 1. The porcine IgM monoclonal antibody of this embodiment was purchased from MP Biomedicals, Incorporated;

[0054] 2. the preparation of horseradish peroxidase (HRP)-anti-PRRSV antibody enzyme conjugate, comprises the steps:

[0055] 1) Routinely immunize rabbits with the purified PRRSV antigen, and when the ELISA titer of the rabbit serum reaches above 1:800, take the serum;

[0056] 2) Precipitating and purifying the serum prepared in step 1) through ammonium sulfate to obtain purified serum;

[0057] 3) HRP-labeling the purified serum prepared in step 2) with an improved sodium periodate oxidation method to obtain a horseradish peroxidase (HRP)-anti-PRRSV antibody enzyme conjugate;

[0058] 4) Add 0.1-10% bovine serum albumin, 0.1-10% casein and 50% neutral glycerol to the enzyme marker (i.e. horseradish peroxidase (HRP)-anti-PRRSV antibo...

Embodiment 2

[0063] Example 2 Detection method of porcine reproductive and respiratory syndrome virus ELISA antibody detection kit

[0064] Utilize the detection method of porcine reproductive and respiratory syndrome virus IgM antibody ELISA detection kit of the present invention, comprise the steps:

[0065] 1) Dilute the sample to be tested 100 times with the sample diluent, add it to the wells of the microplate plate evenly coated with porcine IgM monoclonal antibody, add 100 μl to each well, and set a positive control group, a negative control group and a blank control group at the same time 100 μl positive control solution was added to the positive control group, 100 μl negative control solution was added to the negative control group, 100 μl sample diluent was added to the blank control group, and 3 wells were added in parallel for each sample and control;

[0066] 2) After adding the samples, incubate the ELISA plate at 37° C. for 1 hour, wash the plate with washing solution 3 ti...

Embodiment 3

[0072] Example 3 Specificity and sensitivity test of the kit of the present invention

[0073] 1. Specific detection

[0074] According to the detection method described in Example 2, the kits prepared in Example 1 were used to detect respectively the early serum of porcine PRRSV vaccine immunity, the early serum of porcine PRRSV infection, the purified porcine PRRSV IgG antibody, and the early serum of porcine circovirus type 2 infection , porcine parvovirus antibody positive serum, porcine pseudorabies virus positive serum, swine fever virus antibody positive serum and porcine PRRSV negative serum.

[0075] Dilute the sample to be tested 100 times with the sample diluent, add it to the wells of the microplate plate uniformly coated with 0.1μg-1μg / well of porcine IgM monoclonal antibody, add 100μl to each well, set up a positive control group and a negative control group in parallel 100 μl positive control solution was added to the positive control group, 100 μl negative c...

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Abstract

The invention relates to an ELISA (enzyme-linked immuno sorbent assay) detection kit for porcine reproductive and respiratory syndrome virus IgM (immunoglobulin m) antibodies as well as a preparation method and application of the ELISA detection kit. The ELISA detection kit for the porcine reproductive and respiratory syndrome virus IgM antibodies comprises an ELISA plate coating a porcine IgM monoclonal antibody, sealing fluid, a sample diluent, a detection antigen, an enzyme conjugate, a concentrated cleaning solution, a zymolyte solution A, a zymolyte solution B and a stop solution, wherein the detection antigen is a purified porcine reproductive and respiratory syndrome virus, and the enzyme conjugate is a horse-radish peroxide enzyme-anti-PRRSV (porcine reproductive and respiratory syndrome virus) antibody enzyme conjugate. The specificity of the ELISA detection kit provided by the invention reaches 100% and the sensitivity of the ELISA detection kit reaches up to 1:800, so that the ELISA detection kit can be used for performing early diagnosis on PRRSV infection of a swinery.

Description

technical field [0001] The invention relates to a detection kit, in particular to a porcine reproductive and respiratory syndrome virus (PRRSV) IgM antibody ELISA detection kit, a preparation method and an application thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS), also known as porcine blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) to infect pigs Fever, anorexia, late abortion, premature birth, stillbirth, weak fetus and mummified fetus in pregnant sows, highly contagious disease characterized by respiratory disorders in pigs of all ages (especially piglets). Since it was discovered in 1987, it has spread to all parts of the world and brought great losses to the pig industry. According to reports, PRRS is prevalent in most pig raising areas in China, with an infection rate of over 58%. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577C12N7/02
Inventor 廖园园漆世华李建刘洁秦伟谢红玲温文生
Owner WUHAN CHOPPER BIOLOGY
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