Blocking ELISA antibody detection kit for porcinecircovirus type 2 and preparation method of blocking ELISA antibody detection kit

A porcine circovirus and antibody detection technology, which can be used in measuring devices, instruments, scientific instruments, etc., and can solve problems such as economic losses in the pig industry

Inactive Publication Date: 2017-06-13
JIANGSU NANNONG HI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological and serological surveys have shown that the disease is widespread in many countries and has caused serious economic losses to the pig industry

Method used

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  • Blocking ELISA antibody detection kit for porcinecircovirus type 2 and preparation method of blocking ELISA antibody detection kit
  • Blocking ELISA antibody detection kit for porcinecircovirus type 2 and preparation method of blocking ELISA antibody detection kit
  • Blocking ELISA antibody detection kit for porcinecircovirus type 2 and preparation method of blocking ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] ——Preparation of porcine circovirus type 2 blocking ELISA antibody detection kit

[0138] Taking porcine circovirus type 2 blocking ELISA antibody detection kits 1603001, 1603002, 1603003 as an example

[0139] 1. Preparation and testing of coated antigen

[0140] (1) First-level seed preparation inoculation loop Take an appropriate amount of Escherichia coli (E.coli) recombinant BL-Cap-C strain (CGMCC No.4481) and inoculate it on an LB agar plate containing 50 μg / ml ampicillin. Cultivate statically at 37°C for 24 hours, pick a single typical colony, transfer to 3ml of LB liquid medium containing 50μg / ml ampicillin, and cultivate at 37°C for 12 hours.

[0141] (2) Preparation of secondary seeds Inoculate 2ml of primary seeds (bacteria liquid) into 10ml of LB liquid medium containing 50 μg / ml ampicillin, and culture with shaking at 37°C for 10 hours at 200r / min to obtain secondary seeds.

[0142] (3) Induced expression and purification of the target protein Take 10 ml ...

Embodiment 2

[0205] ——The finished product inspection of a large-scale production of porcine circovirus type 2 blocking ELISA antibody detection kit

[0206] Taking porcine circovirus type 2 blocking ELISA antibody detection kits 1603001, 1603002, 1603003 as an example

[0207] 1. The outer packaging of the trait kit should be clean and undamaged, the label should comply with relevant national regulations, the inner packaging should be undamaged, cracked, and leaked, and the product name, batch number, storage conditions, and expiration date should be clear. in:

[0208] (1) Antigen-coated plate 96-well ELISA plate, packaged in tin foil, well sealed, the bottom of the coated plate is clean and transparent, without cracks, and the capacity is 2 pieces.

[0209] (2) The positive control serum is pale yellow liquid.

[0210] (3) Negative control serum light yellow liquid.

[0211] (4) Enzyme-labeled antibody is a colorless transparent liquid.

[0212] (5) Sample diluent is a colorless tra...

Embodiment 3

[0226] ——Detection method of porcine circovirus type 2 blocking ELISA antibody detection kit

[0227] (1) All reagents should be returned to room temperature (18-25°C) before use. Gently swirl or shake each reagent to make it uniform.

[0228] (2) Washing solution preparation Return the 10-fold concentrated washing solution to room temperature, shake to dissolve the precipitate, then dilute it 10 times with deionized water, and mix well.

[0229] (3) Dilute the serum to be tested 1:1 with the serum diluent in the dilution plate, that is, add 50 μl of the sample diluent to each well, then add 50 μl of the serum to be tested, and use a different tip for each sample. After shaking and mixing, add to each well of the antigen plate. Add positive serum control and negative serum control respectively, 100 μl / well, duplicate 2 wells, and incubate at 37°C for 2 hours.

[0230] (4) Wash and discard the liquid in the wells, add about 300 μl of washing liquid to each well, let stand fo...

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Abstract

The invention relates to a blocking ELISA antibody detection kit for porcinecircovirus type 2 and a preparation method of the blocking ELISA antibody detection kit. The blocking ELISA antibody detection kit for the porcinecircovirus type 2 comprises a coated plate, a PVC2 enzyme labelled antibody, sample diluent, a concentrated washing solution (10*), a positive serum contrast, a negative serum contrast, TMB substrate diluent and a stop solution, wherein the coated plate is prepared from a purified porcine circovirus type 2 nucleocapsid protein CapC protein serving as a non-antigen, and the PVC2 enzyme labelled antibody is prepared from monoclonal antibody hybridoma which is labeled with horse radish peroxidase and secretes PCV2. According to the preparation method, the CapC protein is expressed by virtue of a prokaryotic expression system, and the blocking ELISA is established by taking a monoclonal antibody of PCV2 as the enzyme labelled antibody; and the blocking ELISA antibody detection kit has the characteristics of high specificity and sensitivity and can be applied to the detection of PCV2 antibodies of a porcine serum sample.

Description

technical field [0001] The invention relates to a porcine circovirus type 2 blocking ELISA antibody detection kit and a preparation method thereof, belonging to the field of veterinary biological products. [0002] technical background [0003] Porcine circovirus type 2 (PCV2) is the main pathogen that causes postweaning multisystemic wasting syndrome (PMWS) in weaned piglets. PMWS mainly occurs in weaned piglets aged 5-12 weeks, and its clinical symptoms mainly include progressive emaciation, respiratory symptoms, fever, pallor and jaundice. Etiological and serological investigations showed that the disease is widespread in many countries, causing serious economic losses to the pig industry. [0004] PCV2 belongs to the family Circoviridae, genus Circovirus, and is a single-stranded covalently closed circular DNA virus with a genome of 1766-1768 nt and a simple structure, mainly containing two reading frames (ORFs): ORF1 encodes the virus replication-related protein Rep an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/56983G01N33/531G01N2333/01
Inventor 缪芬芳董彦鹏白娟王先炜姜平王丽敏徐凤胡静雅
Owner JIANGSU NANNONG HI TECH
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