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Alternarin tenuazonic acid artificial antigen, polyclonal antibody, preparation method and application

A technology of gerantoic acid and artificial antigen, applied in the field of analysis and detection, which can solve the problems of inability to produce immunogenicity and small molecular weight, etc.

Inactive Publication Date: 2016-04-20
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular structure of TA is C 9 h 13 o 3 N, with a small molecular weight (M=197), cannot produce immunogenicity by itself. Usually, it is necessary to carry out research on hapten modification and artificial antigen synthesis of TA in order to prepare immune antibodies and then further develop immune products and methods. However, currently on the market There is no commercial TA immune product yet

Method used

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  • Alternarin tenuazonic acid artificial antigen, polyclonal antibody, preparation method and application
  • Alternarin tenuazonic acid artificial antigen, polyclonal antibody, preparation method and application
  • Alternarin tenuazonic acid artificial antigen, polyclonal antibody, preparation method and application

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Embodiment 1

[0027] The synthesis of embodiment 1 hapten TAO

[0028] Take 1.3mg (6.6×10 -3 mmol) TA was dissolved in 1.70 mL of a mixed solvent (methanol: pyridine: water = 1:4:1), and carboxymethyl hydroxylamine hemihydrochloride (H 2 NOCH 2 CO 2 h) 2 - HCl3.3mg, after mixing evenly, add to the rotary evaporator and heat under reflux for 15h, cool to room temperature overnight. The mixture was dried under reduced pressure, 0.30 mL of 2M HCl was added, the composition in the aqueous phase was extracted with dichloromethane as much as possible, the organic phases were combined, dried by adding anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a gum. Purified by dichloromethane / n-hexane reprecipitation to obtain a light brown powder, which was stored at -20°C and detected by HPLC / MS.

[0029]

[0030] Using the hydroxyl group of TA at the C4 position, the hapten was obtained after reacting with carboxymethylhydroxylamine hemihydrochloride, and the stru...

Embodiment 2

[0031] The preparation of embodiment 2 artificial antigen

[0032] The reaction was carried out at a molar ratio of TAO:N-hydroxysuccinimide (NHS):dicyclohexylcarbodiimide (DCC) of 1:5:10. Dissolve TAO in 400 μL dimethylformamide (DMF), add NHS (dissolved in 300 μLDMF), slowly add DCC (dissolved in 300 μLDMF) into a centrifuge tube, and stir overnight at room temperature in the dark with a magnetic stirrer (approx. 16h). Cool the reacted mixture at 4°C for more than 2 hours (if precipitation is found), centrifuge at 10,000 r / min for 5 minutes to obtain a supernatant (active ester solution). TAO-BSA and TAO-KLH were obtained by coupling the active ester solution with BSA and KLH at a feed ratio (60:1, 300:1) respectively, which were purified by dialysis with PBS at 4°C and stored in aliquots.

[0033]

[0034] The last dialyzed external fluid was used as a blank control, and was sucked into a cuvette with TA standard substance, carrier protein solution, hapten solution and...

Embodiment 3

[0037] The preparation of embodiment 3 anti-TA polyclonal antibodies

[0038] Two Blab / c mice were immunized with the prepared immunogen TAO-BSA at doses of 50 μg and 100 μg, respectively, for 5 times: the second immunization was performed 3 weeks after the first immunization, and the immunization was performed every other week thereafter. From the beginning of the second immunization, blood was collected by docking the tail 7 days after each immunization, and the titer of antiserum was detected by indirect non-competition ELISA, and the serum antibody with better immune effect was used for indirect competition ELISA. Eye blood was collected after the last immunization. After each blood collection, the blood was placed in a greenhouse at 37°C for 2 hours, overnight at 4°C, centrifuged at 8000r / min, an equal volume of glycerol was added to the supernatant and mixed evenly, and stored in a -20°C refrigerator for later use.

[0039] 20μg / mLTAO-KLH was used as the coating source ...

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Abstract

The invention discloses an alternarin tenuazonic acid artificial antigen, a polyclonal antibody, a preparation method and application, and particularly discloses an artificial antigen obtained by coupling a compound (formula I) serving as a tenuazonic acid hapten, a hapten TAO, bovine serum albumin, hemocyanin and other macromolecular protein, a polyclonal antibody prepared from the artificial antigen and application of the artificial antigen and the polyclonal antibody to tenuazonic acid detection. TA (tenuazonic acid) can be detected by setting up an indirect competitive ELISA method, a theoretical basis is provided for a TA immunological detection method, a foundation is laid for further developing and detecting TA immunology fast detection products, and a new path is provided for setting up the TA immunological detection method, developing the TA immunological series products, and fast and efficiently monitoring TA contamination conditions.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to an artificial antigen of alternaria mycotoxin alternaria alternaria ketoacid, a polyclonal antibody, a preparation method and application thereof. Background technique [0002] Alternaria tenuazonic acid (Tenuazonicacid, TA) is one of the toxic metabolites produced by Alternaria, Magnaporthe oryzae and other molds under specific conditions, and has been found in the United States, Canada, Brazil, China and other countries. TA can extensively contaminate food and feed, lead to post-harvest rot of fruits, grains and vegetables, and can cause stem point mildew and rice blast in parasitic and saprophytic plants. Among the more than 70 known Alternaria mycotoxins with obvious toxicity, TA is the most toxic and the pollution level ranks first among all Alternaria mycotoxins. TA has a variety of biological properties, such as inhibiting protein synthesis by i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D207/38C07K14/765C07K14/795C07K16/14G01N33/53G01N33/531
CPCC07D207/38C07K14/765C07K14/795C07K16/14C07K19/00G01N33/53G01N33/531
Inventor 马良张宇昊吴春生钟红
Owner SOUTHWEST UNIVERSITY
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