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Quantitative detection kit for porcine circovirus type 2 antigen

A technology for quantitative detection of porcine circovirus, applied in the field of biotechnology detection, can solve the problems of high cost, poor broad-spectrum recognition, and poor consistency of results, and achieve the effects of short operation cycle, good specificity, and high coincidence rate

Active Publication Date: 2017-08-11
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former has the problems of poor stability between batches and poor consistency of results; in terms of cost, since the preparation of the kit requires the production of multiple antibodies, the production method is more complicated and the cost is higher
The kit constructed in the latter way cannot meet the demand in terms of detection sensitivity
[0005] In addition, in practical applications, it is found that commercially available monoclonal antibodies often have poor broad-spectrum recognition of different PCV2 strains used by various manufacturers, and the detection work relies on expensive imported PCV2 monoclonal antibody or polyantibody detection serum

Method used

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  • Quantitative detection kit for porcine circovirus type 2 antigen
  • Quantitative detection kit for porcine circovirus type 2 antigen
  • Quantitative detection kit for porcine circovirus type 2 antigen

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0031] Example 1 Preparation of immune antigen

[0032] In order to maintain the natural structure of the immune antigen (PCV2-NJ strain), improve the purification efficiency, and shorten the purification cycle, a nickel column was used to mount the recombinant protein His-VHH for specific affinity purification of the PCV2-NJ strain virus.

[0033] His-VHH is an anti-PCV2 nanobody with His-tagged protein. The preparation method of the recombinant protein is as follows: the nucleoside of the anti-PCV2 nanobody VHH in the patent No. ZL201210249621.4 and the name "anti-porcine circovirus type 2 Bactrian camel heavy chain single domain antibody and its preparation method and use" NdeI and HindIII enzyme cutting sites were added to the upstream and downstream of the acid sequence (see SEQ ID No.4 in the patent sequence list), and the DNA fragment was artificially synthesized. The synthetic fragment was inserted between the NdeI and HindIII restriction sites of the expression vector ...

example 2

[0035] Example 2 Screening, Identification, Preparation and Purification of Monoclonal Antibodies

[0036] 1. Mice Immunization

[0037] The PCV2-NJ strain antigen obtained in Example 1 was used as the immunization antigen, and the protein content was detected using the BCA protein quantification kit (purchased from Pierce Company), the concentration was adjusted to 0.5 μg / μL, and the 6-week-old was immunized by subcutaneous injection at multiple points on the back of the neck. For female Balb / c mice, the first immunization dose was 100 μg / mouse, and three weeks later, the dose was boosted with 50 μg / mouse, and then the booster immunization was carried out again at intervals of 2 weeks. Blood was collected one week after the last immunization, and the serum titer of immunized mice was detected by indirect ELISA method with non-immunized mouse serum as a control.

[0038] The indirect ELISA method is as follows: the mouse serum is diluted 10-fold with PBST buffer, and the dilu...

example 3

[0062] Example 3 Preparation of enzyme-labeled antibody

[0063] 1. Preparation of horseradish peroxidase-labeled anti-PCV2 monoclonal antibody

[0064] Anti-PCV2 monoclonal antibodies 1B2', 1G9', 4C2', and 23F5' were labeled with horseradish peroxidase, and the specific methods were as follows:

[0065] (1) Dissolve 5mg HRP (horseradish peroxidase) in 1mL deionized water, add 0.2ml, 0.1M NaIO 4 solution, mix well, airtight, and react at room temperature for 20 minutes in the dark.

[0066] (2) Put the solution obtained in step (1) into a dialysis bag, and dialyze overnight at 4° C. with pH 4.4, 1 mM sodium acetate buffer.

[0067] (3) 10 mg of the purified monoclonal antibody 1B2', 1G9', 4C2' or 23F5' was dialyzed overnight with pH 9.5, 10 mM carbonate buffer.

[0068] (4) Take the solution in the dialysis bag after the dialysis in step (2), add 40ul pH9.5, 0.2M carbonate buffer to adjust the pH to 9.0-9.5, and then add the monoclonal antibody after (3) treatment, Mix wel...

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Abstract

The invention provides a quantitative detection kit for a porcine circovirus type 2 antigen, belonging to the field of biotechnological detection. A hybridoma cell strain 1G9 secreting a monoclonal antibody against the porcine circovirus type 2 has an accession number of CCTCC No. C2016105. The quantitative detection kit for the porcine circovirus type 2 antigen comprises a detection antibody and an ELISA plated coated with the monoclonal antibody against the porcine circovirus type 2, wherein the detection antibody is an enzyme-labeled monoclonal antibody against the porcine circovirus type 2. The quantitative detection kit for the porcine circovirus type 2 antigen in the invention only uses one antibody as a coated and detection antibody, and is simple to prepare, low in cost, good in broad spectrum activity of the antigen, and high in sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a porcine circovirus type 2 antigen quantitative detection kit. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) belongs to Circoviridae Circovirus genus, is a non-enveloped single-strand negative-sense circular DNA virus with a diameter of 17-20nm, and is the smallest known animal virus one. According to the coding gene of porcine circovirus type 2 structural protein Cap, it can be subdivided into multiple genotypes, such as PCV2a, PCV2b, PCV2c, etc. Among them, PCV2b, which plays a major pathogenic role, has multiple strains. PCV2 infection can cause multisystemic wasting syndrome in weaned piglets, reproductive failure in pregnant sows, respiratory disease in weaned pigs and finishing pigs, porcine dermatitis and nephritic syndrome, congenital tremor in young piglets and other diseases. PCV2 infection was successively discovered...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535G01N33/569
CPCG01N33/535G01N33/56983G01N33/577G01N2333/01
Inventor 张浩明杨利郑其升陈瑾乔绪稳侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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