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Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application

A porcine circovirus, monoclonal antibody technology, applied in antiviral immunoglobulin, measuring devices, instruments, etc., can solve the problems of high cost, not suitable for clinical diagnosis, rapid, popular application, long diagnosis cycle, etc. Stable, high potency and powerful effect

Inactive Publication Date: 2010-07-07
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, many scholars at home and abroad have established a variety of diagnostic methods to detect porcine circovirus type 2 and its antibodies, such as polymerase chain reaction (PCR) technology for detection of pathogens, in situ hybridization methods, immunohistochemical methods, etc. and detection Antibody indirect immunofluorescence technology, enzyme-linked immunosorbent assay (ELISA) method, etc., but these diagnostic methods have limitations such as long diagnostic cycle and high cost, and are not suitable for rapid and popular application in clinical diagnosis

Method used

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  • Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application
  • Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application
  • Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] A kind of preparation method of porcine circovirus type II protein monoclonal antibody:

[0030] 1. Materials and methods

[0031] 1.1 Strains, cells and experimental animals

[0032] PCV2 isolates, PK15 cell lines, and myeloma cells SP2 / 0 were all preserved by the Animal Disease Department of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, and BALB / c mice were purchased from Fujian College of Traditional Chinese Medicine.

[0033] 1.2 Main reagents

[0034] HAT medium, HT medium, fetal calf serum, DMEM high glucose medium, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, and fluorescein (FITC)-labeled goat anti-mouse IgG were all purchased from Sigma.

[0035] 1.3 Antigen preparation

[0036] The cryopreserved PCV2 was resuscitated and expanded for culture, 55,000r·min -1 Centrifuge for 4 hours, dissolve with an appropriate amount of PBS, collect the precipitate, and lyse it with ultrasonic waves. The mas...

Embodiment 2

[0061] This example is the porcine circovirus type II protein monoclonal antibody prepared in Example 1.

Embodiment 3

[0063] The application of the porcine circovirus type II protein monoclonal antibody prepared by embodiment 1 is:

[0064] 1 Making slices of disease materials Take suspected PCV2 disease materials, cut out soybean-sized lung and lymph node tissue blocks, place them on the tissue fixer and freeze them. After the tissue blocks are frozen, put them on the microtome. For thin tissue slices of mm, fix the glass slides soaked in cold ethanol in cold acetone, take them out to dry, and store them at -20°C for later use.

[0065] 2 Establishment of indirect immunofluorescence diagnostic method Take out the prepared disease material slices, add PCV2 mouse positive serum, 8-60 and 10-48 culture supernatant monoclonal antibody respectively, put them into a sealed bag and incubate at 37°C for 1 hour, take out and use PBS -T washing for 5 min, a total of 3 times, and then dropwise adding different concentrations (1:30000, 1:20000, 1:10000) of goat anti-mouse fluorescent secondary antibody,...

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Abstract

The invention discloses a preparation method for a PCV-II Cap protein monoclonal antibody, an antibody and application. The invention adopts ultracentrifuged and purified PCV-II as an immunogen to immunize a BALB / c mouse by the conventional method, takes spleen cells of the immunized BALB / c mouse to fuse with SP2 / 0 cells, obtains two strains of hybridoma cells secreting the PCV2-Cap protein monoclonal antibodies by indirect ELISA screening, respectively names the two strains of hybridoma cells as 8-60 and 10-48, identifies biological characteristics of the two strains 8-60 and 10-48, and usesthe two strains 8-60 and 10-48 as the first antibodies to establish an indirect immunofluorescence diagnostic method. The result of the indirect immunofluorescence diagnostic method is basically consistent with that of the PCR diagnostic method, and the positive and negative coincidence rates are respectively 93.75 percent and 100 percent so as to provide reference for preventing and treating theporcine circovirus disease.

Description

technical field [0001] The invention relates to a porcine circovirus type II Cap protein monoclonal antibody, in particular to a preparation method of the porcine circovirus type II Cap protein monoclonal antibody, the antibody and application thereof. Background technique [0002] In recent years, post-weaning multisystemic wasting syndrome (Post-weaning multisystemic wasting syndrome, PMWS) is a pig disease characterized by fever, progressive emaciation, respiratory and digestive system disorders, and currently exists in many countries and regions. The prevalence of the disease has caused huge economic losses to the pig industry. Porcine circovirus type 2 (PCV2) is considered to be the main pathogen of PMWS, and it is also associated with many other pig diseases, such as porcine respiratory syndrome and porcine dermatitis nephrotic syndrome. [0003] At present, many scholars at home and abroad have established a variety of diagnostic methods to detect porcine circovirus ...

Claims

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Application Information

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IPC IPC(8): C07K16/08G01N33/577G01N21/64
Inventor 车勇良周伦江庄向生魏宏王隆柏陈少莺陈如敬
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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