Porcine circovirus type 2 antigen capture ELISA kit

A technology of porcine circovirus and kit, applied in the field of immunology, can solve the problems of time-consuming operation, poor specificity, and high technical requirements, and achieve the effect of short time-consuming and simple operation

Active Publication Date: 2015-07-01
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the PCR technique has the highest sensitivity, poor specificity, and poor repeatability; the method for determining the toxicity is high in specificity, and is also a commonly used method for isolating and identifying the virus at present, but this method is time-consuming and requires high technical requirements for the operator; Although capture ELISA is not as sensitive as the above methods, it has high specificity and is easy to operate, saving time and effort

Method used

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  • Porcine circovirus type 2 antigen capture ELISA kit
  • Porcine circovirus type 2 antigen capture ELISA kit
  • Porcine circovirus type 2 antigen capture ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of monoclonal antibody against porcine circovirus type 2 (PCV2) Cap protein

[0056] 1. Materials and methods

[0057] 1.1 Cells, virus species, experimental animals and reagents

[0058] SP2 / 0 and PK15 cell lines, PCV1 / G strains, PCV2 strains (LG, JF, JF2, CL, YJ, SH, BDH, AH, etc.) are all preserved by the Porcine Circovirus Research Group of Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences ; 6-week-old female BALB / c mice were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HAT and HT mixed salt and PEG1450 were purchased from Sigma; pre-stained protein Marker was purchased from Fermentas; Mouse MonoAb-ID Kit( HRP), HRP-Goat Anti-Mouse IgG (H+L) were purchased from Invitrogen; other reagents are domestically pure.

[0059] 1.2 Purification of Porcine Circovirus Type 2 (PCV2)

[0060] The PCV2a / LG strain was purified by CsCl gradient density cent...

Embodiment 2

[0082] Example 2 Preparation of polyclonal antibodies against porcine circovirus type 2 (PCV2)

[0083] 1 Materials and methods

[0084] 1.1 Test materials

[0085] 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) Preserved by the Porcine Circovirus Research Group of Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences, 6 piglets 30-40 days old (PCV2-IPMA serum antibody titer is less than or equal to 1:50 times) by Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences Provided by the Laboratory Animal Center.

[0086] 1.2 Test method

[0087] 1.2.1 Animal immunity test

[0088] Six piglets were randomly divided into a test group and a control group, with 3 pigs in each group and reared separately. In the first immunization, piglets were injected with 1 mL of inactivated porcine circovirus type 2 (LG strain) vaccine into th...

Embodiment 3

[0102] Example 3 Establishment of an ELISA method for capturing porcine circovirus type 2 antigen

[0103] 1. Materials and methods

[0104] 1.1 Material

[0105] ABTS was purchased from BBI, DMEM medium and fetal bovine serum (FBS) were purchased from Gibco, and other reagents were domestically pure.

[0106] Preparation of ABTS working solution: (1) Chromogenic A solution (0.1mol / L citrate buffer) preparation: weigh 29.41g sodium citrate (Na 3 C 6 H 5 O 7 ·2H 2 O) Dissolve in 1800mL deionized water, adjust the pH to 4.3 with citric acid, and then add 200μL 30% H 2 O 2 , And finally dilute the volume to 2000mL with deionized water; (2) Preparation of color developing solution B (2.19% ABTS solution): weigh 2.19g ABTS powder, fully dissolve it with 0.1mol / L citrate buffer, and dilute to 100mL; (3) Preparation of ABTS working solution: add 0.5ml of chromogenic A solution to 9.5mL of chromogenic A solution, mix well and use, ready to use.

[0107] 1.2 monoclonal antibody purification and...

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Abstract

The invention discloses a porcine circovirus type 2 antigen capture ELISA kit. The kit internally comprises an enzyme labeled monoclonal antibody which is secreted by a hybridoma cell strain with the preservation number of CGMCC NO.10205. The invention also discloses a capture ELISA method for rapidly detecting the porcine circovirus type 2 and established by utilizing the monoclonal antibody. A porcine circovirus type 2 polyclonal antibody and a porcine circovirus type 2 cap protein monoclonal antibody respectively act as a capture antibody and a detecting antibody. The method can be used for detecting multiple gene type PCV2 viruses; the detection sensibility is 400 TICD50 / ml; the porcine circovirus type 2 has no crossed reaction with other swine viruses, and the toxic value determination method coincidence rate is 88%. The result shows that the method has the advantages of operation simplicity, good specificity, high sensitiveness, short time consumption and the like, can be used for estimating the toxic value of viruses, and can conveniently and rapidly control the quality of PCV2 inactivated vaccine semi-finished products.

Description

Technical field [0001] The invention relates to a kit, in particular to a porcine circovirus type 2 antigen capture ELISA kit, which belongs to the field of immunology. Background technique [0002] Porcine circovirus type 2 (PCV2) belongs to the circovirus genus of the Circoviridae family. It is one of the smallest animal viruses so far, with a diameter of 17 nm, icosahedral symmetry, and no capsule. The viral genome is composed of a single strand of closed circular DNA, containing 1766~1769 nucleotides (Olvera A, Cortey M, Segalés J. Molecular evolution of porcine circovirus type 2 genomes: Phylogeny and clonality[J].Virology,2007,357( 2):175~185; Huang LP,Lu YH,Wei YW,et al.Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2.BMC Microbiol.2011a,11:188. ). The PCV2 genome mainly contains two large open reading frames (ORF1 and ORF2) (Cheung A K. Transcriptional analysis of porcine c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 黄立平危艳武刘长明
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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