Monoclonal antibody of tenuazonic acid and its enzyme-linked immunosorbent assay method

A technology of Alternaria alpendorf acid and Alternaria necrotic acid monoclonal, which is applied in the field of monoclonal antibody and enzyme-linked immunoassay detection of Alternaria alpenoids acid, which can solve the problem of large capital investment and time-consuming , the operation process is cumbersome and other problems, to achieve the effect of low cost, good correlation and good specificity

Inactive Publication Date: 2017-10-17
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the instrument detection takes a lot of time, the operation process is cumbersome, and it also requires a large capital investment, so it is difficult to meet the on-site screening needs of a large number of samples
[0005] In addition, there are not many reports on the rapid detection of TEA at present, and most of the detection objects are derivatives of TEA, the derivation process is relatively cumbersome, which brings a lot of inconvenience to the detection of actual samples

Method used

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  • Monoclonal antibody of tenuazonic acid and its enzyme-linked immunosorbent assay method
  • Monoclonal antibody of tenuazonic acid and its enzyme-linked immunosorbent assay method
  • Monoclonal antibody of tenuazonic acid and its enzyme-linked immunosorbent assay method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation and Characterization of Alternarinic Acid Immunogen

[0055] 1. Preparation of alternarinic acid hapten

[0056] 50mg alternarinic acid (TEA, Mw=197.1) and 50mg carboxymethylhydroxylamine hemihydrochloride (CMO, Mw=109.3) were dissolved in 4.0mL, 2.0mL mixed solvent (methanol:pyridine:water =1:4:1), then slowly add the dissolved CMO solution into the dissolved TEA solution, mix evenly, place in an oil bath and heat under reflux at 70°C for 15h. After the reaction (monitor whether the TEA is completely reacted, if not, the reaction can be continued), the mixture is dried under reduced pressure, and 3.0 mL of 2 mol / L HCl solution is added to dissolve the dry matter. The compound in the aqueous phase was extracted several times with dichloromethane, and the organic phases were combined and concentrated under reduced pressure to obtain a light brown oil. Purification by dichloromethane / n-hexane reprecipitation gave a light brown powder, which was the...

Embodiment 2

[0063] The preparation of embodiment 2 alternarinic acid monoclonal antibody

[0064] 1. Animal immunity

[0065] When 6-month-old female Balb / C mice were immunized for the first time, the amount of immunogen injected was 0.1 mL / mouse, and 1 mg / mL of immunogen was added with an equal volume of Freund's complete adjuvant, and the fully emulsified liquid was placed in a small Inject subcutaneously at multiple points on the back and abdomen of the mouse, about 200 μl per point; perform the second immunization 21 days later, the immunization dose is 0.1 mL / rat, and emulsify with Freund's incomplete adjuvant; after that, boost the immunization twice every 14 days, and generally co-immunize 5 times. In this experiment, after the third immunization and the fifth immunization, blood was collected 7 days later for antiserum titer determination. Three days before the cell fusion, the mice were boosted by direct intraperitoneal injection of 0.1 mL of the immunogen (without adjuvant). ...

Embodiment 3

[0087] Example 3 Mass Preparation of Alternarinic Acid Monoclonal Antibody

[0088] 1. Antibody secretion and expression

[0089] After the hybridoma cell clones secreting specific monoclonal antibodies are obtained, monoclonal antibodies are usually produced in large quantities by in vitro culture and in vivo induction of monoclonal antibodies in animals. The conventional method is: inject liquid stone mole intraperitoneally in more than a dozen Balb / c mice over 8 weeks old in advance, at a dose of 0.5 mL / mouse, and inject hybridoma cells into the peritoneal cavity of the mice 1 to 2 weeks later. . Observe the state of the mice every day after cell inoculation, especially from the seventh day onwards, the spirit of the mice will appear sluggish, and the abdominal cavity will swell. At this time, it is necessary to pay close attention to the state of the mice. Collect ascites aseptically, centrifuge the collected ascites at 12000r / min for 10 min, remove the upper layer of fa...

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Abstract

The invention discloses a monoclonal antibody capable of specifically identifying tenuazonic acid and its enzyme-linked immunosorbent assay method. The monoclonal antibody of tenuazonic acid is prepared by firstly preparing hapten (TEA-CMO), and then respectively coupling hemocyanin (KLH) and bovine serum albumin (BSA) to obtain immunogen (TEA-CMO-KLH) and package (TEA-CMO-BSA); then applying animal immunization and hybridoma technique to obtain the monoclonal antibody of tenuazonic acid; an indirect competition enzyme-linked immunosorbent assay method built by the monoclonal antibody of tenuazonic acid is a rapid detecting method of tenuazonic acid residue; the detection limit (IC10) of the method is 1.00 ng/mL, IC50=18.50 ng/mL; the linear detection scale is 3.56-96.24 ng/mL; the direct detection of the tenuazonic acid can be realized; the monoclonal antibody is free from crossing reaction with similar matters of another nine alternaria alternata aflatoxin structures; the method is good in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of toxin detection. More specifically, it relates to a monoclonal antibody to alternarinic acid and an ELISA detection method thereof. Background technique [0002] Alternaria tenuazonic acid (Tenuazonic Acid, TEA) is one of the main toxic metabolites of Alternaria, and its molecular structure is C 9 h 13 o 3 N (as shown in Table 2), the molecular weight is 197. TEA is listed as a toxic chemical by the U.S. Food and Drug Administration (FDA). Registry of Toxic Effects of Chemical Substance ") (Yang Xin, 2000). Alternaria mycotoxins cause widespread contamination of commercial crops and food worldwide, among which TEA is found in melons, apples, apple products, oranges, olives, green peppers, red peppers, tomatoes, tomato products, sunflower seeds, sorghum, wheat, edible Foods such as oil (including olive oil, rapeseed oil, sesame oil, and sunflower oil) are highly polluted. At present, more than 70 Al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 王弘周晓文沈玉栋王锋徐振林孙远明肖治理杨金易
Owner SOUTH CHINA AGRI UNIV
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