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Porcine circovirus type 2 competitive ELISA antibody detection kit

A technology of porcine circovirus and kit, which is applied in the field of immunology, can solve the problems of limitation, difficult high-throughput and automatic detection, and low sensitivity, and achieves the effect of simple method, automatic detection requirements, and good activity

Inactive Publication Date: 2016-01-06
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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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 type 2 competitive ELISA antibody detection kit
  • Porcine circovirus type 2 competitive ELISA antibody detection kit
  • Porcine circovirus type 2 competitive 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. The 1st immunization, piglet neck intramuscular injection porcine circovirus type 2 (LG strain) inactivated vaccine 1mL" only -1 Carry out immunization for the second time at an interval of 21 days, and the immunization method and dosage are the same as above; ...

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 type 2 (PCV2) competitive ELISA antibody detection kit. The kit includes an enzyme-labeled monoclonal antibody secreted by a hybridoma cell strain having the preservation number of CGMCC NO.10205. The invention also discloses a competitive ELISA method established by using the monoclonal antibody and used for detecting a porcine serum PCV2 antibody. The method comprises that firstly an ELISA plate is wrapped with a PCV2 positive serum to capture a PCV2 antigen, then the PCV2 antigen undergoes a reaction with a to-be-detected porcine serum antibody and an HRP-labeled mono-antibody 3A5, and a competitive ELISA detection result is showed through measuring the enzyme-labeled mono-antibody. The method and an IPMA method perform parallel tests on 237 porcine serum samples, the two methods have the detecting coincidence rate being 94.1%, and the method provided by the invention has the sensitivity of 92.6% and the specificity of 98.4%, and has no cross reaction with other porcine virus reference positive serums. The kit has the characteristics of simple operation, good specificity, high sensitivity and the like, and provides an effective technical means 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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IPC IPC(8): G01N33/577G01N33/569
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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