Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method

A technology for a quantitative detection method for Clostridium perfringens, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of inability to perform quantitative and large-scale detection, expensive detection kits, and not particularly high sensitivity, and achieves shortened time. Detection cycle, detection speed, and specificity are good.

Active Publication Date: 2014-07-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

However, the sensitivity of these methods is not particularly high, and the operability in China is lacking, and the detection kits available abroad are expensive and cannot be quantitatively and mass-produced, so it is difficult to promote in China. Therefore, in order to make up for this method in domestic α toxin detection Therefore, there is an urgent need to establish a fast, sensitive, specific ELISA method capable of quantitative and large-scale detection. The establishment of this method also provides an effective tool for early diagnosis and follow-up research of Clostridium perfringens

Method used

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  • Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method
  • Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method
  • Clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method

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Embodiment 1

[0034] Example 1 Prokaryotic expression of Clostridium perfringens alpha toxin

[0035] Design specific primers according to primmer5 software:

[0036] Upstream primer: 5'-GCGGAATTCATGAAAAGAAAGATTTGT-3', as shown in SEQ No.1;

[0037] Downstream primer: 5'-GCGGCGAAGCTTTTATTTTATATTATAAGTT-3', as shown in SEQ No.2;

[0038] After enrichment of the standard strain NCTC528 was selected, the DNA was extracted with a bacterial genome DNA extraction kit, and then PCR was used to amplify the α-toxin gene fragment of Clostridium perfringens. Amplification conditions: 94°C for 5min, 94°C for 1min, 54°C for 1min , 72°C for 70s, a total of 32 cycles, 72°C extension for 7min. The target product purified by the DNA purification and recovery kit was connected to pMD18-T and sequenced. The correctly sequenced recombinant plasmid and vector pET-28a were digested with EcoR I and Xho I respectively, and then transferred into expression competent cells BL21 after ligation, and the bacterial s...

Embodiment 2

[0039] Example 2 Preparation of Anti-Clostridium perfringens Alpha Toxin Monoclonal Antibody

[0040] Select pure female BALB / c mice (4-6 weeks old) homologous to myeloma cell Sp2 / 0 as immunized animals, immunization scheme: the first immunization is the recombinant α-toxin protein prepared in step 1 and an equal volume of Freund’s After complete emulsification of the complete adjuvant, 100 μg / intraperitoneal injection; two weeks and five weeks after the first immunization for the second and third immunizations, respectively, after complete emulsification of recombinant α-toxin protein and an equal volume of Freund’s incomplete adjuvant, 100 μg / intraperitoneal injection Injection: Three weeks after the third immunization, 50-100 μg of recombinant α-toxin protein without adjuvant was injected intraperitoneally, and the splenocytes of the mouse with the highest titer were collected 3-5 days after the injection for the next step of cell fusion. During the period, blood was collec...

Embodiment 3

[0042] Example 3 Preparation of polyclonal antibody against Clostridium perfringens alpha toxin:

[0043] Select 5 New Zealand white rabbits of 2-3Kg, the first immunization is the purified recombinant α-toxin and an equal volume of Freund's complete adjuvant after complete emulsification, multi-point subcutaneous injection of 1 mg / rabbit on the back, and then Freund's incomplete adjuvant and equal volume of purified Recombinant α-toxin was completely emulsified and immunized once every 2 weeks. After 3 immunizations, purified recombinant α-toxin without adjuvant was used to boost immunization 2 weeks later. Blood was collected from rabbit heart to obtain serum after 15 days. The antibody was purified by saturated ammonium sulfate precipitation method to obtain polyclonal antibody against Clostridium perfringens α-toxin. The antibody titer was detected by indirect ELISA method, and the purity was detected by SDS-PAGE electrophoresis.

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Abstract

The invention relates to a clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method. According to the clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method, alpha toxin protein obtained via prokaryotic expression is taken as an immunogen; monoclonal antibodies obtained via hybridoma technique are taken as detection antibodies and capture antibodies; reaction conditions are optimized via experiments; alpha toxin protein samples with a series of concentration are used for construction of a standard curve; the double-antibody sandwich ELIS method is established; and indexes of the double-antibody sandwich ELIS are verified. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method specifically comprises following steps: (1) prokaryotic expression of alpha toxin; (2) preparation of anti-alpha toxin monoclonal antibodies; (3) establishment of the double-antibody sandwich ELIS method; (4) establishment of the standard curve; and (5) performance evaluation on the double-antibody sandwich ELIS method. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method is excellent in specificity, and high in sensitivity and stability, is fast and convenient, and can be used for effective quantitative determination of alpha toxin in A-E type clostridium perfringens cultural supernatants; and the high-efficient detection method is provided for alpha toxin determination.

Description

(1) Technical field [0001] The invention relates to a quantitative detection method of Clostridium perfringens alpha toxin double-antibody sandwich ELISA. (2) Background of the invention [0002] Clostridium perfringens can cause lamb dysentery, necrotic enteritis, enterotoxemia and other diseases in lambs, calves, piglets, rabbits, chicks, etc., with acute onset and high mortality, and is an important disease that seriously endangers the breeding industry. Bring huge economic losses to the development of animal husbandry in various countries. All types of Clostridium perfringens produce α-toxin (CPA), so α-toxin is an important basis for diagnosing Clostridioides welmannii disease and an important indicator for judging food safety. [0003] The classic methods for detecting α-toxin include animal test, toxin neutralization test, lecithin decomposition test, etc., but the operation is cumbersome and the sensitivity is low. In recent years, with the development of biotechno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/56911G01N33/577
Inventor 王海荣孙佳芝柴同杰李课陈勇逄伟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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