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Indirect ELISA detection method of clostridium perfringens type A toxin antibody

A Clostridium perfringens and detection method technology, applied in the field of animal bacteriology, can solve the problems of insensitivity, large serum samples and animals, cumbersome operation, etc., and achieve consistent results, strong specificity, and good repeatability Effect

Active Publication Date: 2016-11-02
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

The bacteria can infect a variety of animals, causing diseases such as necrotic enteritis and enterotoxemia, and bring serious economic losses to the breeding industry
[0003] The antibody level of Clostridium perfringens toxin is an index to evaluate the resistance of animals to Clostridium perfringens disease. Toxin neutralization test in mice is a classic method to detect antibody to Clostridium perfringens type A, although it is accurate and reliable , but time-consuming and labor-intensive, the amount of serum samples and animals is relatively large, and the results may be inaccurate due to individual differences in animals; it is also difficult to meet the needs of practice in rapid diagnosis
However, the lecithin hydrolysis inhibition test is not sensitive, has poor repeatability, is cumbersome to operate, and is difficult to popularize and apply.
Indirect hemagglutination test requires the preparation of hemagglutination antigen, which is relatively complicated and difficult to popularize in production practice
These methods are limited by many conditions, and it is difficult to adapt to the current control of the epidemic of Clostridium perfringens and the needs of research and detection of the disease

Method used

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  • Indirect ELISA detection method of clostridium perfringens type A toxin antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The preparation of embodiment 1 type A Clostridium perfringens exotoxin:

[0042] Type A Clostridium perfringens strains (National Collection of Type Cultures-British Microorganism Collection Center Preservation Number: NCTC528) were inoculated on the blood plate medium for recovery, anaerobic culture at 37°C for 36h, and a single Colony into Thioglycollate Nutrient Broth Enrichment Medium in a gas concentration of 88% N 2 , 7%H 2 , 5%CO 2Anaerobic culture at 38°C for 12 h under anaerobic conditions. Inoculate 5mL type A Clostridium perfringens enrichment liquid into 100ml pH7.5 toxin-producing medium (dissolve 2g peptone, 1g dextrin, 2g yeast extract and 1.2g L-arginine per 100mL PBS buffer , 1g glucose), shake culture in an anaerobic environment, culture at 43°C for 5h to efficiently produce toxin, then centrifuge at 8000r / min at 4°C for 15min, and then filter and sterilize with a Chua filter with a pore size of 0.22μm to obtain the sterilized Type A exotoxin of C...

Embodiment 2

[0043] Example 2 Concentration, Purification Coated Antigen and Determination of its Concentration

[0044] Slowly add the sterilized toxin solution into saturated ammonium sulfate solution until the volume concentration of ammonium sulfate in the mixed solution reaches 50%, and let stand at 4°C overnight; the next day, centrifuge the solution at 8000r / min for 20min, discard the supernatant, Dissolve the precipitate with an appropriate amount of 0.05M Tris-HCL buffer solution, slowly add saturated ammonium sulfate solution to make the final concentration of ammonium sulfate reach 40%, repeat the above operation, and finally dissolve the precipitate in Tris-HCL; then use Sephadex-G25 column layer The exotoxin was analyzed and desalted, purified and concentrated; the purified toxin protein was subjected to SDS-PAGE electrophoresis to analyze the protein purity, and the results showed that there was an obvious band at the molecular weight of 43KD (such as figure 1 Shown), is the ...

Embodiment 3

[0045] Example 3 Immunization of rabbits, preparation of positive serum

[0046] Add 0.3% formaldehyde to the exotoxin solution, mix thoroughly, inactivate at 37°C for 96 hours, and shake once every 5-6 hours during this period.

[0047] Select 3 healthy unvaccinated rabbits of 1.0-1.5Kg, mix and emulsify the above-mentioned inactivated Clostridium perfringens type A exotoxin and Freund's complete adjuvant according to volume 1:1, and inject into the back skin at multiple points ; 7 days and 15 days after the first immunization, the above-mentioned inactivated Clostridium perfringens type A exotoxin and Freund's complete adjuvant were mixed and emulsified according to the volume 1:1, and the rabbits were immunized for the second and third immunizations; After 10 days, the rabbits were culled, the blood was collected aseptically, coagulated naturally, and the supernatant was collected by centrifugation. The obtained hyperimmune serum was used as positive control serum for indir...

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Abstract

The present invention relates to the field of animal bacteriology, and provides an indirect ELISA detection method of a clostridium perfringens type A toxin antibody. The method uses prepared clostridium perfringens type A toxin as an antigen; the antigen is used for immunizing rabbit to prepare a hyperimmune serum, and is also concentrated and purified as a coating antigen for indirect ELISA. The detection method includes the following steps: (1) coating; (2) enclosing; (3) adding a sample to be tested; (4) adding a secondary antibody; (5) developing; (6) terminating; and (7) determining the results. The method has the advantages of high specificity, strong sensitivity, good repeatability and low cost. In addition, the method can detect samples in a large amount, and provides an effective and simple serological diagnosis method for the detection and study of the disease.

Description

technical field [0001] The invention relates to the field of animal bacteriology and provides an indirect ELISA detection method for type A Clostridium perfringens toxin antibody. Background technique [0002] Clostridium perfringens (C.perfringens), also known as Clostridium welchii (C.welchii), is an opportunistic pathogen that parasitizes in the gastrointestinal tract of humans and animals under normal conditions. Clostridium perfringens is divided into five subtypes A, B, C, D, and E according to the four main exotoxins (α, β, ε, ι) secreted by it. The main pathogenic exotoxin secreted by Clostridium perfringens type A is alpha toxin. Alpha toxin is a multifunctional metalloenzyme dependent on zinc ions, which can destroy the phospholipids on the surface of the host cell membrane, thereby destroying the function of the cell membrane. The bacterium can infect a variety of animals, causing diseases such as necrotic enteritis and enterotoxemia, and causing serious economi...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/543G01N2333/33
Inventor 王海荣林晓佳柴同杰陈长俊
Owner SHANDONG AGRICULTURAL UNIVERSITY
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