ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for porcine circovirus type 2 IgM antibody and preparation method of ELISA detection kit

A porcine circovirus and detection kit technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problem of rarely detecting PCV2IgM antibodies

Inactive Publication Date: 2013-09-18
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 PCV2 or ant...

Method used

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  • ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for porcine circovirus type 2 IgM antibody and preparation method of ELISA detection kit
  • ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for porcine circovirus type 2 IgM antibody and preparation method of ELISA detection kit
  • ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for porcine circovirus type 2 IgM antibody and preparation method of ELISA detection kit

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0060] Example 1 Preparation of ELISA kit for detection of porcine circovirus type 2 IgM antibody

[0061] 1. The porcine IgM monoclonal antibody of this embodiment was purchased from MP Biomedicals, Incorporated;

[0062] 2. the preparation of horseradish peroxidase (HRP)-anti-Cap protein antibody enzyme conjugate, comprises the following steps:

[0063] 1) Routinely immunize rabbits with the purified PCV2-Cap protein, and when the ELISA titer of the rabbit serum reaches 1:1000 or more, take the serum;

[0064] 2) Precipitating and purifying the serum prepared in step 1) through ammonium sulfate to obtain purified serum;

[0065] 3) HRP-labeling the purified serum prepared in step 2) with an improved sodium periodate oxidation method to obtain a horseradish peroxidase (HRP)-anti-Cap protein antibody enzyme conjugate;

[0066] 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-Cap prot...

Embodiment 2

[0079] Example 2Detection method of porcine circovirus type 2 ELISA antibody detection kit

[0080] Utilize the detection method of porcine circovirus type 2 IgM antibody ELISA detection kit of the present invention, comprise the steps:

[0081] 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;

[0082] 2) After adding the samples, incubate the ELISA plate at 37° C. for 1 hour, wash the plate with washing solution 3 times, and spin dry. The washing solution is prepared b...

Embodiment 3

[0088] Example 3 Specificity and sensitivity test of the kit of the present invention

[0089] 1. Specific detection

[0090] 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 PCV2 inactivated vaccine immunity, the early serum of porcine PCV2 infection, the purified porcine PCV2 IgG antibody, the early serum of porcine PCV1 infection, and the porcine PCV1 infection early serum. PRRS virus antibody positive serum, porcine pseudorabies virus positive serum, swine fever virus antibody positive serum and porcine PCV2 negative serum.

[0091] 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 cont...

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Abstract

The invention relates to an ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for a porcine circovirus type 2 IgM antibody as well as a preparation method and application of the ELISA detection kit. The ELISA detection kit for the porcine circovirus type 2 IgM antibody comprises an ELISA plate for coating a pig IgM monoclonal antibody, a confining liquid, a sample diluent, an antigen for detection, an enzyme conjugate, a concentrated washing liquid, an enzyme substrate solution A, an enzyme substrate solution B and a stop solution, wherein the antigen for detection is a purified porcine circovirus type 2 nucleocapsid protein (PCV2-Cap protein) antigen, and the enzyme conjugate is a horse radish peroxidase-antiCap protein antibody enzyme conjugate. The kit provided by the invention can realize 100% of specificity expression, has the high sensitivity of 1:800, and can be used for the early diagnosis of PCV2 infection of a swinery.

Description

technical field [0001] The invention relates to a detection kit, in particular to a porcine circovirus type 2 IgM antibody ELISA detection kit, a detection method and an application thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes postweaning multisystemic wasting syndrome (PMWS) in weaned piglets. In addition to causing PMWS, PCV2 is also associated with congenital tremor in piglets, porcine dermatitis nephritic syndrome, porcine respiratory syndrome (PDNS), and reproductive disorders in sows. Since the first discovery of PMWS in Canada in 1996, the associated diseases caused by PCV2 infection have been widespread all over the world. Up to now, the harm of PCV2 infection in my country's pig industry has become increasingly prominent. Therefore, the clinical demand for the development of PCV2 serum antibody detection kits is increasingly urgent, and the detection of porcine circovirus-specific IgM antibodies provides ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577
Inventor 廖园园漆世华李建秦伟朱薇谢红玲温文生
Owner WUHAN CHOPPER BIOLOGY
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