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Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof

A technology for isolating alternaria and artificial antigens, which is applied in the field of artificial antigens, antibodies and their preparation, and haptens for intersecting alternaria, which can solve the problems of rapid screening, time-consuming and labor-intensive, and high operation requirements that are not suitable for large quantities problems, to achieve high accuracy, broad application prospects, and good sensitivity

Active Publication Date: 2016-06-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the limit standard of Alternaria toxin in crops and food has not been established at home and abroad, and there are few reports on immunoassay methods. Traditional detection methods such as gas chromatography, gas chromatography-mass spectrometry , liquid chromatography-mass spectrometry and capillary electrophoresis are not only time-consuming and labor-intensive, but also require high operating requirements, which are not suitable for rapid screening in large quantities. Therefore, it is urgent to invent a fast and simple detection method for Alternaria toxin

Method used

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  • Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof
  • Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof
  • Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Preparation method of hapten H1 (n=1)

[0046] In a 25mL round bottom flask, 260mg (1mmol) alternatinol was dissolved in 3mL anhydrous DMF, and 170.6mg (1.2mmol) K was added under magnetic stirring. 2 CO 3 , then added 200.4mg (1.5mmol) ethyl bromoacetate dropwise, reacted at room temperature for 4h, added 1M HCl to terminate the reaction, the reaction mixture was purified by silica gel column, eluted with ethyl acetate: n-hexane = 1:5, spin Drying gave the intermediate product as a white powder. Then dissolve it in tetrahydrofuran by volume ratio: H 2Add 100mgLiOH to the solution of O=1:1, hydrolyze for 12h, adjust the pH=3~4 with 1M HCl, extract 5 times with ethyl acetate, concentrate and purify by silica gel column, use methanol: chloroform: glacial acetic acid=1:30: After elution at 0.01, the eluate was spin-dried to obtain a brownish-yellow powder, which was the target hapten, see formula (I).

[0047] , where n=1.

Embodiment 2

[0048] The preparation of embodiment 2 immunogen / coating original

[0049] In the preparation method of the immunogen and the coating source, the difference lies in the type of carrier protein used, the immunogen carrier protein mainly adopts bovine serum albumin (BSA), and the coating original carrier protein mainly adopts egg white protein (OVA ), the coupling method used is the active ester method. The preparation method of the immunogen described below is an example.

[0050] Active ester method: Dissolve hapten AOH-C 5 mg (0.015 mol) in 0.3 mL DMSO, add 4.78 mg DCC (0.023 mol), 2.65 mg NHS (0.023 mol), stir overnight at 4°C, this is solution A; take 12.3 mg bovine serum The protein was dissolved in 6 mL of PBS buffer solution with pH=7.4, solution A was added dropwise, reacted overnight at 4°C, centrifuged to take the supernatant, dialyzed with normal saline for 3 days, and changed the dialysate 4 times a day to obtain Alternaria alternata phenol immunogen; in addition,...

Embodiment 3

[0054] Example 3 Antibody Preparation and Identification

[0055] Emulsify the prepared immunogen with the same amount of immune adjuvant (incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) and immunize animals. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles, and ear veins. The second immunization was performed 4 weeks later, and the immunization was added every 3 weeks thereafter. One week after the fourth booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 10,000°C for 15 minutes at 4°C, and the supernatant was taken as antiserum. The antise...

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Abstract

The invention belongs to the technical field of food safety immunodetection and particularly discloses hapten, artificial antigen and antibody directly targeted to alternariol and a preparation method and application thereof. The alternariol hapten is of the structure as indicated in formula (I), wherein n=1, 2. The alternariol artificial antigen is of the structure as indicated in formula (III), wherein n=1, 2; carrier protein is keyhole limpet haemocyanin or bovine serum albumin or ovalbumin. The titer of antiserum obtained from an immune animal of the artificial antigen can reach 1: 128000, the minimum detection limit is 1.15 ng / mL, the half inhibiting concentration is 16.5 ng / mL, the antibody has the remarkable advantages of being high in specificity, good in sensitivity, high in accuracy and the like, therefore the antigen and the antibody can be used for establishing an alternariol enzyme linked immunosorbenption and analysis technology, and thereby the hapten, artificial antigen and antibody directly targeted to alternariol and the preparation method and application thereof can be used for rapidly detecting residual alternariol in food and have wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, and more specifically relates to a hapten, an artificial antigen, an antibody and a preparation method and application thereof that are intersected with alternaria. Background technique [0002] Altemariol ((Altemariol, AOH) is a class of small molecular compounds with mutagenicity, carcinogenicity, genotoxicity and other toxicities. It also belongs to the diphenylpyrone derivatives and is one of the toxins of the secondary metabolites of Alternaria. K.Wollenhaupt et al. studied the effect of AOH on the activity of pig endometrial cells. When the amount of AOH added was 12.5 μmol / L, the number of endometrial cells was significantly reduced; when the concentration of AOH was 3.12μmol / L, the metabolic activity of the cells was significantly affected; Pollock et al. studied the subacute toxicity and teratogenic effects of AOH and AME, when 200mg / L When a concentration of 8 kg was i...

Claims

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Application Information

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IPC IPC(8): C07D311/80C07K14/765C07K14/77C07K14/795C07K1/107G01N33/543
CPCC07D311/80C07K1/1077C07K14/765C07K14/77C07K14/795C07K19/00G01N33/543
Inventor 沈玉栋朱帆王弘杨金易徐振林肖治理孙远明雷红涛
Owner SOUTH CHINA AGRI UNIV
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