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Capture ELISA (enzyme-linked immunosorbent assay) detection method for Clostridium perfringens Alpha-toxin antibody

A technology of Clostridium perfringens and a detection method, which is applied in the field of animal bacteriology, can solve the problems of insensitivity, high price, labor-intensive, etc., and achieves the effect of improving accuracy and specificity, and strong specificity.

Inactive Publication Date: 2017-06-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, the mouse toxin neutralization test is a classic method for detecting Clostridium perfringens toxin antibody. Although it is accurate and reliable, it is time-consuming and labor-intensive.
However, the lecithin hydrolysis inhibition test is not sensitive, has poor repeatability, is cumbersome to operate, and is difficult to popularize and apply.
In recent years, with the development of biotechnology, a variety of commodity detection kits for the detection of Clostridium perfringens toxin antibodies have appeared abroad, but they are expensive. There is an urgent need to establish a rapid, sensitive, specific and large-scale detection ELISA method to provide an effective tool for the diagnosis and research of Clostridium perfringens
[0004] The inventor of the present invention once applied for a patent: "An Indirect ELISA Detection Method for Clostridium perfringens Type A Toxin Antibody", although the indirect ELISA detection method for the Clostridium perfringens Type A Toxin Antibody is disclosed, However, because it detects all exotoxins of Clostridium perfringens type A, it has poor specificity and cannot qualitatively detect Clostridium perfringens alpha toxin, so it is difficult to fill the gap in the prior art

Method used

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  • Capture ELISA (enzyme-linked immunosorbent assay) detection method for Clostridium perfringens Alpha-toxin antibody
  • Capture ELISA (enzyme-linked immunosorbent assay) detection method for Clostridium perfringens Alpha-toxin antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0055] Preparation and purification of Example 1A type Clostridium perfringens exotoxin

[0056] 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 2 Anaerobic 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 exot...

Embodiment 2

[0058] Example 2 Recovery of anti-Clostridium perfringens α-toxin hybridoma cells and preparation and purification of monoclonal antibodies;

[0059] The hybridoma cell line F12 of Clostridium perfringens α-toxin (preserved in China Microorganism Culture Collection, preservation number: CGMCC No.8870) was routinely recovered and cultured; 6-8 weeks old female BALB / c 10 mice were sensitized by intraperitoneal injection of 0.5 mL of Freund's incomplete adjuvant, and injected with positive hybridoma cell line F12 one week later, at a dose of 0.5-1×10 6 One per mouse, the mouse ascites was collected after 7-10 days, and a large amount of Clostridium perfringens α-toxin monoclonal antibody was obtained. The ascites was purified by n-octanoic acid-ammonium sulfate method, and the purification effect was detected by SDS-PAGE. The results are as follows figure 2 Shown: Two obvious protein bands can be seen in the samples in lanes 1 and 2 in the figure: a heavy chain of about 53kD an...

Embodiment 3

[0060] Example 3 Preparation of Anti-Clostridium perfringens Alpha Toxin Polyclonal Antibody

[0061] Add formaldehyde to the exotoxin of Clostridium perfringens type A obtained in Example 1 after sterilization to make the final concentration 0.3vt%, after fully mixing, inactivate at 37°C for 96h, during which every 5-6h Oscillate shake once;

[0062] Select 5 healthy rabbits of 1.0-1.5Kg who have not been vaccinated, mix and emulsify the inactivated α-toxin 2mL and Freund's complete adjuvant at a volume of 1:1, and inject 1mg / rabbit at multiple points on the back skin; One week later, mix and emulsify inactivated exotoxin 2mL and Freund's incomplete adjuvant at a volume of 1:1, and the injection dose and method are the same as above; 2 weeks later, the same method is used for the third immunization; Antigen booster immunization, the dose and method are the same as above, the obtained hyperimmune serum was used as the capture ELISA positive control serum; the control group wa...

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Abstract

The invention relates to the field of animal bacteriology and provides a capture ELISA (enzyme-linked immunosorbent assay) detection method for a Clostridium perfringens Alpha-toxin antibody; in the method, prepared type-A Clostridium perfringens exotoxin is used as an antigen that is used for immunizing a rabbit to obtain rabbit serum and for acting as a binding antigen for capture ELISA after being concentrated and purified; the detection method comprises the steps of (1) enveloping; (2) sealing; (3) adding an antigen; (4) adding a sample to be tested; (5) adding a secondary antibody; (6) developing color; (7) ending; (8) judging results. The detection method has the advantages of high specificity, high sensitivity, good repeatability, low cost and the like; in addition, the detection method is suitable for detecting samples in large batch, and an effective and simple serological diagnostic method is provided for the detection and research on such toxin.

Description

technical field [0001] The invention relates to the field of animal bacteriology and provides an ELISA detection method for Clostridium perfringens alpha toxin antibody capture. Background technique [0002] Clostridium perfringens (C.perfringens), also known as Clostridium welchii (C.welchii), widely exists in the natural environment, and is found in the digestive tract of almost all warm-blooded animals, and belongs to the normal intestinal flora of humans and animals a member of. Clostridium perfringens can cause lamb dysentery, necrotic enteritis and enterotoxemia in lambs, calves, piglets, rabbits and chicks. In recent years, the main pathogen of livestock "sudden death syndrome" in my country has brought huge economic losses to the development of animal husbandry in various countries. All types of Clostridium perfringens produce α-toxin (CPA). This bacterium can cause gas gangrene in humans and enterotoxemia and necrotic enteritis in various animals. When livestock ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 王海荣楚国茹柴同杰钟召兵李超陈勇林晓佳王方山
Owner SHANDONG AGRICULTURAL UNIVERSITY
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