Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin

A technology of Clostridium perfringens and monoclonal antibody, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inability to quantify and mass detection, the high price of detection kits, and the sensitivity is not particularly high, and achieve detection High speed, high sensitivity and good specificity

Inactive Publication Date: 2015-04-15
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods lack operability in China, and the sensitivity is not particularly high, and the detection kits available abroad are expensive, cannot be quantitatively and mass-produced, and are less likely to be popularized in China. Therefore, in order to fill the domestic α toxin detection gap Blank, need to establish a rapid, sensitive, specific ELISA method capable of quantitative and large-scale detection

Method used

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  • Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin
  • Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin
  • Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Prokaryotic expression of Clostridium perfringens alpha toxin:

[0042] According to the published gene sequence of Clostridium perfringens alpha toxin in GenBank (accession number CP 000246.1), design primers P1 and P2:

[0043] P1: 5'-GGGG GAATTCTGGGATGGAAA-3' (the underline indicates that the introduced enzyme cleavage site is EcoR I).

[0044] P2: 5'-GGGG CTCGAG TTATTTTATAT-3' (the underline indicates that the introduced enzyme cleavage site is Xho I).

[0045] Select the standard strain ATCC13124, extract the DNA with a bacterial genome DNA extraction kit, and then use PCR to amplify the α-toxin gene fragment of Clostridium perfringens. Amplification conditions: pre-denaturation at 94°C for 10 minutes, denaturation at 94°C for 30 seconds, and annealing at 50.2°C A total of 30 cycles of 30 s, 72°C extension for 1 min, 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 49.6°C annealing for 30 s, 72°C extension for 1 min, and a final extension at 72°C ...

Embodiment 2

[0047] Preparation of polyclonal antibody against Clostridium perfringens alpha toxin:

[0048] Select 2-3Kg New Zealand white rabbits, 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 on the back, the dose is 2mg / rabbit, two weeks after the first immunization, Freund's incomplete adjuvant is used The dose is 1 mg / rat, immunized once a week, and one week after 3 immunizations, the purified recombinant α-toxin without adjuvant is used to boost the immunization. After 7 days, blood is collected from the heart of the rabbit to obtain serum. Anti-Clostridium perfringens α-toxin polyclonal antibody, the antibody titer was detected by indirect ELISA method, and Western blotting was used for the identification of polyclonal antibody characteristics.

Embodiment 3

[0050] Preparation of monoclonal antibody against Clostridium perfringens alpha toxin:

[0051] Pure female BALB / c mice (4-6 weeks old) homologous to myeloma cell SP2 / 0 were selected as immunized animals. Immunization scheme: α-toxin recombinant protein (obtained in step 1) after gel-excision purification and renaturation ) to prepare monoclonal antibodies for the immunogen. For the first immunization, 50 μg of purified protein was emulsified with an equal volume of complete Freund’s adjuvant, and immunized 6-week-old female BALB / c mice by intraperitoneal injection; after 3 weeks, 50 μg of purified protein was mixed with an equal volume of incomplete Freund’s adjuvant Dose emulsification for the second immunization; after that, immunization was carried out every 2 weeks, the immunization dose and immunization route were the same as the second immunization, and 3-5 days after the last immunization, the mouse splenocytes with the highest titer were used for cell fusion.

[0052...

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Abstract

The invention discloses a monoclonal antibody blocking ELISA testing method of a clostridium perfringens alpha toxin, which is used for early testing of the alpha toxin and provides an effective testing tool for early diagnosis, spreading prevention and subsequent research of clostridium perfringens. The method comprises the following two steps: I, establishing the monoclonal antibody blocking ELISA testing method; and II, determining a result judgment criterion. The monoclonal antibody blocking ELISA testing method which has good specificity and stability and high sensitivity, is quick, simple and convenient, and tests the alpha toxin efficiently is established for the first time, and the method is simple to implement and high in testing speed and can achieve quantitative testing and massive testing. In a word, monoclonal antibody blocking ELISA is expected to play an important role in prevention and treatment of clostridium perfringens diseases.

Description

technical field [0001] The invention relates to a monoclonal antibody blocking ELISA method for detecting alpha toxin of Clostridium perfringens. Background technique [0002] Clostridium perfringens is a normal flora in the digestive tract of humans and animals, and is the main pathogen that causes traumatic gas gangrene, food poisoning, necrotic enteritis and enterotoxemia in humans. In poultry, it mainly causes necrotic enteritis and endangers the poultry industry. In livestock, it mainly causes "sudden death syndrome" in cattle and sheep, lamb dysentery, enterotoxemia in cattle and soft kidney disease in sheep. Alpha toxin (CPA) is a multifunctional metalloenzyme that can be produced by almost all types of Clostridium perfringens, and is a typical lethal toxin. Therefore, alpha toxin is an important basis for the diagnosis of Clostridium perfringens. It is an important indicator for judging food safety. [0003] The classic methods for detecting α-toxin include toxin ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/577
Inventor 李广兴乔艺然张艳郑晓星任玉东
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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