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Porcine circovirus II competition ELISA antibody detection kit

A porcine circovirus and kit technology, applied in the field of immunology, can solve the problems of low sensitivity, limitation, difficulty in high-throughput and automatic detection, etc., and achieve the effects of good activity, simple method, and automatic detection requirements.

Active Publication Date: 2016-03-30
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The methods used for PCV2 serum antibody detection are mainly IIF, IPMA, indirect ELISA, blocking ELISA, competitive ELISA and immune colloidal gold technology, etc., among which IIF and IPMA are the most classic and commonly used. Although these two methods have many advantages, but in There is a certain degree of subjectivity in the judgment of the results, and it is difficult to achieve high-throughput and automatic detection, which limits its promotion
The indirect ELISA based on recombinant protein has good specificity, but its sensitivity is lower than that of IPMA (Zhang Zhaoxia, Liu Changming, Wei Yanwu, et al. Development and application of ELISA antibody detection kit for porcine circovirus type 2[J] . Chinese Journal of Preventive Veterinary Medicine, 2008, 30(7): 548-551; Zhang Zhihui, Wei Yanwu, Wu Hongli, etc. Comparison and application of several ELISA antibody detection kits for porcine circovirus type 2[J]. Journal of Veterinary Medicine, 2014, 36(2): 129-133), the test results of the above methods cannot directly reflect PCV2 neutralizing antibodies

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  • Porcine circovirus II competition ELISA antibody detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation of embodiment 1 anti-porcine circovirus type 2 (PCV2) Cap protein monoclonal antibody

[0053] 1. Materials and methods

[0054] 1.1 Cells, virus species, experimental animals, antibodies and reagents

[0055] SP2 / 0 and PK15 cell lines (PCV1 and PCV2 negative), PCV1 / G strains, PCV2 strains (LG, JF, JF2, CL, YJ, SH, BDH, AH, etc.) Preserved by the porcine circovirus research group; 6-week-old female BALB / c mice were provided by the Experimental Animal Center of the same institute. ABTS was purchased from BBI Company, HAT and HT mixed salt, PEG1450, 3-amino-9-ethylimidazole (AEC), horseradish peroxidase (HRP, TypeVI-A) and HRP-labeled rabbit anti-mouse IgG (H+ L) were purchased from Sigma; DMEM medium and calf serum (FBS) were purchased from Gibco; prestained protein markers were purchased from Fermentas; MouseMonoAb-IDKit (HRP), HRP-GoatAnti-MouseIgG (H+L) Purchased from Invitrogen Company; horseradish peroxidase (HRP)-ProteinA marker (HRP-SPA) is Zyme...

Embodiment 2

[0083] The preparation of embodiment 2 porcine circovirus type 2 (PCV2) positive serum and negative serum

[0084] 1 Materials and methods

[0085] 1.1 Test material

[0086] Porcine circovirus type 2 (LG strain) inactivated vaccine was provided by Harbin Veken Biotechnology Development Company, PCV2 / YJ strain (10 5.0 TCID 50 / ml) was preserved by the porcine circovirus research group of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Provided by Experimental Animal Center.

[0087] 1.2 Test method

[0088] 1.2.1 Animal immunity test

[0089] Six piglets were randomly divided into experimental group and control group, with 3 piglets in each group, and were fed separately. For the first immunization, piglets were intramuscularly injected with porcine circovirus type 2 (LG strain) inactivated vaccine 1mL per piglet -1 ; The second immunization was carried out at an interval of 21 days, and the immunization method and dose were the...

Embodiment 3

[0103] The establishment of embodiment 3 porcine circovirus type 2 competitive ELISA method

[0104] 1. Materials and methods

[0105] 1.1 Materials

[0106] PCV2 negative and positive sera were self-preserved by our laboratory, and reference positive sera of several other porcine viruses were provided by relevant research groups of our institute. 3-amino-9-ethylimidazole (AEC) and horseradish peroxidase (HRP, TypeVI-A) are Sigma products; ABTS) is a BBI product; horseradish peroxidase (HRP)-ProteinA marker (HRP-SPA) is a Zymed product; ProteinG chromatography column is a GE Healthcare product; DMEM medium and fetal bovine serum (FBS) are purchased from Gibco Company; other reagents were of domestic analytical grade.

[0107] Preparation of ABTS working solution: (1) preparation of chromogenic A solution (0.1mol / L citrate buffer): weigh 29.41g of sodium citrate (Na 3 C 6 h 5 o 7 2H 2 O) Dissolve in 1800 mL of deionized water, adjust the pH to 4.3 with citric acid, and ...

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Abstract

The invention discloses a porcine circovirus II competition ELISA antibody detection kit. The kit contains a peroxidase labeled monoclonal antibody secreted by a hybridoma cell strain with the preservation No. being CGMCC No.10205. The invention also discloses a method for utilizing the monoclonal antibody to establish a competition ELISA for detecting porcine serum PCV2 antibody. The method comprises the following steps: coating ELISA plate with PCV2 positive serum, thereby capturing PCV2 antigen, and then reacting with to-be-detected porcine serum antibody and HRP-marked monoclonal antibody 3A5 and displaying a competition ELISA detection result by measuring the peroxidase labeled monoclonal antibody. The method and the IPMA method are utilized to perform parallel test on 237 porcine serum samples; the detection coincidence rate is 94.1% according to the two methods; the sensibility is 92.6% and the specificity is 98.4% according to the method; the kit has no cross reaction with other porcine circovirus reference positive serums; the kit provided by the invention has the characteristics of simple and easy operation, high specificity, high sensibility, and the like; an effective technical method is supplied for PCV2 epidemiological investigation and serum antibody detection.

Description

technical field [0001] The invention relates to a kit, in particular to a porcine circovirus type 2 competitive ELISA antibody detection kit, which belongs to the field of immunology. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) is a member of the genus Circovirus in the Circoviridae family. It is one of the smallest viruses among animal viruses so far, with a diameter of 17 nm, icosahedral symmetry, and no envelope. The viral genome consists of a single strand of closed circular DNA, containing 1766-1769 nucleotides (OlveraA, CorteyM, SegalésJ. Molecularevolutionofporcinecircovirustype2genomes: Phylogenyandclonality[J]. Virology, 2007, 357(2): 175-185; HuangL.P., LuY.H., WeiY.W., et al. Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circular virus type 2. BMC Microbiol. 2011a, 11: 188.). The PCV2 genome mainly contains two large open reading frames...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/01
Inventor 黄立平刘长明危艳武朱海波
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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