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PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof

A detection method and a virus-like technology, which is applied in the field of animal disease diagnosis and biological products, can solve the problems of low detection sensitivity, inapplicability, and lack of confirmation by sandwich ELISA, and achieve the effect of high sensitivity and good quantitative accuracy

Active Publication Date: 2020-03-17
JIANGSU NANNONG HI TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the PCV2 Cap does not form VLPs, it cannot enter the cell. After the PCV2 Cap is assembled to form VLPs, the VLPs can enter the cell. Then, indirect immunofluorescence and other related operations are performed. If the cells show a positive signal, it can be judged that the cell is in the cell. Contains PCV2 VLPs, the method can identify and determine the presence of VLPs, but is not suitable for quantitative analysis
[0005] In terms of PCV2 VLPs quantification, there is currently no effective means to distinguish the content of VLPs and Cap, and the detection sensitivity is low. Generally, the results obtained by sandwich ELISA quantification It is the overall concentration after mixing VLPs and Cap, and it is impossible to quantify VLPs of different genotypes of PCV2 at the same time. There is a lack of a sandwich ELISA method that can distinguish VLPs and Cap and quantify VLPs in different genotypes of PCV2
In the description of the existing patent (porcine circovirus type 2 double antibody sandwich ELISA kit and its application, application number 201811095919.8), the PCV2 Cap protein is used to prepare monoclonal antibodies, and the monoclonal antibodies do not recognize a single subclass of Cap in Western Blot. Based on linear epitopes, double-antibody sandwich ELISA can be used to quantify PCV2 semi-finished inactivated vaccines, and the minimum detection value is 3 μg / ml. However, since VLPs subunit vaccines are assembled in vitro by Cap proteins, the Cap proteins contained in them The impact on quantification cannot be ruled out, and it is not applicable to the quantification of PCV2 VLPs subunit vaccines
In the description of another existing patent (Monoclonal Antibody Recognizing PCV2 Virus-Like Particles and Its Application in Qualitative and Quantitative Detection of PCV2 Virus-like Particles, Application No. 201811261025.1), the monoclonal antibody was prepared by preparing PCV2 VLPs. Anti-linear epitopes that do not recognize a single subunit of Cap in Western Blot, using indirect ELISA to prove that the contained monoclonal antibodies only have specific antigen-antibody reactions with correctly assembled VLPs, but did not use sandwich ELISA to confirm whether they can distinguish between VLPs and Cap protein, the lowest detection limit of double-antibody sandwich ELISA established by using this monoclonal antibody is 146.48ng / ml

Method used

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  • PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof
  • PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof
  • PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1——Expression of PCV2d Cap protein

[0071] 1) Transform the pET100-PCV 2d-SS-Cap-Pro recombinant expression plasmid into BL21(DE3) Escherichia coli (Beijing Quanshijin Company) competent cells (recombinant Escherichia coli porcine circovirus 2d type Cap gene (pET100-PCV2d -SS-Cap-Pro) strain, which was sent to the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences on December 28, 2017, the address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 15135) was coated on an LB medium plate with ampicillin at a concentration of 100 μg / mL, and cultured upside down at 37° C. for 12 to 14 hours.

[0072] 2) Randomly pick a monoclonal colony to 10 mL of LB medium containing 100 μg / mL ampicillin antibiotic, and shake and culture at 220 r / min at 37° C. for 12 hours.

[0073] 3) According to the ratio of (V / V) 1:100, add 10 mL of bacterial solution to 1 L of α-lactose fermentati...

Embodiment 2

[0074] Embodiment 2——purification of PCV 2d Cap protein by affinity chromatography

[0075] 1) The Escherichia coli precipitate is placed on an ice bath, and 100mL of PBS lysis buffer (Lysis buffer, its formula is: lysis buffer: NaCl 500mM, NaCl 2 HPO 4 100mM, Imidazole 30mM, KH 2 PO 4 100mM, Triton X-1001% (freshly added), pH 8.0) to lyse the centrifuged precipitate of 1L culture solution (concentrated according to the volume ratio of 1:10 times), and fully suspend the bacterial precipitate.

[0076] 2) The suspended sample is crushed and precipitated by a low-temperature ultra-high pressure continuous flow cell crusher, and the pressure is 1300bar, and the crushing is repeated 6 to 8 times. The crushed product is stained by Gram staining method, and more than 95% of E. coli are completely crushed under a microscope. The samples were then centrifuged at 16,000 g for 20 min at 4°C, and the supernatant was collected after centrifugation.

[0077] 3) Wash the Ni-NTA filler...

Embodiment 3

[0087] Example 3 - Assembly of PCV 2d virus-like particles

[0088] After performing SDS-PAGE analysis on the protein purified by affinity chromatography prepared in Example 2, select a high-concentration and high-purity protein and place it in a 7000D size dialysis bag and place it in a 4°C freezer in the assembly buffer of the present invention (Ammoniumcitrate 200mM, Na 3 PO 4 100mM, Tris 20mM, Arginine 20mM, pH7.0) in dialysis for 48h, the samples collected after dialysis were negatively stained with 1% phosphotungstic acid, and the shape, size and concentration of virus-like particles observed under a transmission electron microscope were basically the same (see image 3 ).

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Abstract

The invention relates to a PCV2 virus-like particle sandwich quantitative ELISA detection method and application thereof. Porcine circovirus type II PCV2d VLPs immunizing mice are used for screening hybridoma cells; the prepared antibody can specifically recognize PCV2, wherein the PCV2b and PCV2d can be recognized; and the antibody can be applied to PCV2 related detection of WB, IFA and the like.When the antibody is used for coating in ELISA detection, the antibody does not react with Cap protein and can be used for distinguishing VLPs and Cap. The sandwich ELISA is established through optimization of a series of reaction conditions according to the monoclonal antibody and can be used for specific quantification and qualitative determination of PCV2 VLPs; the lowest detection concentration can reach 62.5 ng / ml, the sensitivity is high, the correlation coefficient R2 is 0.992, and the quantification accuracy is high. The PCV2 virus-like particle sandwich quantitative ELISA detection method is suitable for quantification of PCV2b and PCV2d VLPs and can be applied to quantification and assembly efficiency identification of different genotypes of VLPs of PCV2 and PCV2 virus infectionidentification.

Description

technical field [0001] The invention relates to a PCVd virus-like particle sandwich quantitative ELISA detection method and application thereof, belonging to the field of animal disease diagnosis and biological products. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen of porcine dermatitis nephrotic syndrome, multisystemic wasting syndrome and reproductive failure syndrome in weaned piglets, and has an important economic impact on the pig industry. The PCV2 genome is about 1.7Kb, which belongs to the single-stranded positive-strand circular DNA virus, and contains two main open reading frames, ORF2 encodes the viral nucleocapsid protein (Cap), and ORF1 encodes the Rep protein related to viral replication. The viral nucleocapsid (Capsid) is an icosahedron composed of 60 Cap protein subunits, without an envelope, and its particle diameter is about 17nm. [0003] At present, vaccines are used as a preventive method for prevention and control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577C07K16/08C12N5/20
CPCG01N33/56983G01N33/577C07K16/08G01N2333/01C07K2317/33
Inventor 王乃东邹亚文杨毅王东亮湛洋杨文兵董彦鹏于浩洋
Owner JIANGSU NANNONG HI TECH
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