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Antibody capturing ELISA detection method for C.perfringens beta toxin

A technology of Clostridium perfringens and β-toxin, which is applied in the field of animal immunology, can solve the problems of poor exposure and display of antigenic determinants, and achieve the effect of rapid response, good sensitivity and high sensitivity

Active Publication Date: 2019-09-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method uses prokaryotic expressed recombinant protein as the antigen, the exposure and display of the epitope is poor, and the detection sensitivity of the β-toxin antibody needs to be further improved

Method used

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  • Antibody capturing ELISA detection method for C.perfringens beta toxin
  • Antibody capturing ELISA detection method for C.perfringens beta toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation and purification of exotoxin of Clostridium perfringens type C

[0055] C-type Clostridium perfringens strains (purchased from the National Collection of Type Cultures (National Collection of Type Cultures), preservation number: NCTC3180) were inoculated on a blood plate medium for resuscitation and cultured anaerobic at 37°C for 36 hours. Pick a single colony into the thioglycolate nutrient broth enrichment medium, where the gas concentration is 88%N 2 , 7% H 2 , 5% CO 2 Anaerobic culture was conducted at 38°C for 12h under anaerobic environment. Inoculate 5mL C-type Clostridium perfringens bacteria enrichment liquid into 100ml pH 7.5 toxin production medium (each 100mL PBS buffer solution dissolves 2g peptone, 1g dextrin, 2g yeast extract and 1.2g L-arginine , 1g glucose), shake culture in an anaerobic environment, culture at 43°C for 5h to efficiently produce toxins, then centrifuge at 8000r / min at 4°C for 15min, then filter and sterilize in a Chu...

Embodiment 2

[0057] Example 2: Resuscitation of anti-C. perfringens beta toxin hybridoma cells and preparation and purification of monoclonal antibodies;

[0058] The hybridoma cell line 2C8A of Clostridium perfringens β-toxin (preserved in the China Collection of Microorganisms, accession number: CGMCC NO.14894) was routinely resuscitated and cultured; female BALB / c cells aged 6-8 weeks were selected 10 mice were sensitized by intraperitoneal injection of 0.5 mL of Freund's incomplete adjuvant, and the positive hybridoma cell line 2C8A was injected one week later, the dose was 0.5-1×10 6 Each mouse ascites was collected 7-10 days later to obtain a large number of clostridium perfringens beta toxin monoclonal antibodies. The ascites was purified by the n-octanoic acid-ammonium sulfate method, and the purification effect was tested by SDS-PAGE. The results are as follows figure 2 As shown, the samples in lanes 1 and 2 in the figure can see two obvious protein bands: a heavy chain of about 53kD...

Embodiment 3

[0059] Example 3: Preparation of polyclonal antibody against Clostridium perfringens beta toxin

[0060] Add formaldehyde to the exotoxin of Clostridium perfringens type C after sterilization obtained in Example 1 to make the final concentration 0.3vt%, and after mixing thoroughly, inactivate at 37°C for 96h, during which every 5-6h Shake once;

[0061] Choose 5 healthy and unvaccinated rabbits of 1.0-1.5Kg, mix and emulsify the inactivated beta toxin 2mL and Freund's complete adjuvant in a volume of 1:1, and multiple injections of 1mg per rabbit on the back skin; After one week, 2mL of inactivated exotoxin and Freund’s incomplete adjuvant were mixed and emulsified in a volume of 1:1, and the injection dosage and method were the same as above; two weeks later, the same method was used for three immunizations; two weeks after three immunizations, no adjuvant was used Antigen booster immunization, the dosage and method are the same as above, the obtained hyperimmune serum is used as...

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Abstract

The invention relates to an antibody capturing ELISA detection method for C.perfringens beta toxin, and belongs to the technical field of animal immunology. A monoclonal antibody of the C.perfringens beta toxin coats an enzyme label plate, natural C.perfringens beta toxin serves as a capturing antigen, and the antibody capturing ELISA detection method for C.perfringens beta toxin is thus established. The method is characterized by being rapid, simple, highly-specific and highly sensitive, and suitable for the aspects including C.perfringens beta toxin detection, epidemiologic investigation and vaccine immunization effect evaluation.

Description

Technical field [0001] The present invention relates to the technical field of animal immunology, in particular to a capture ELISA detection method for Clostridium perfringens beta toxin antibody. Background technique [0002] Clostridium perfringens (C.perfringens), also known as Clostridium welchii (C.welchii), is widely found in the natural environment and found in the digestive tract of almost all warm-blooded animals. It belongs to the normal flora of human and animal intestines the member of. Clostridium perfringens can cause lamb dysentery and necrotizing enteritis and enterotoxemia of lambs, calves, piglets, rabbits, and chicks. It has acute onset and high mortality. It is an important disease that seriously harms the breeding industry. In recent years, the main pathogen of the "sudden death syndrome" of livestock in our country has brought huge economic losses to the development of animal husbandry in various countries. Type C Clostridium perfringens mainly produces β ...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/577
CPCG01N33/558G01N33/577
Inventor 王海荣陈端楚国茹柴同杰陈勇钟招兵
Owner SHANDONG AGRICULTURAL UNIVERSITY
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