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Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen

A technology of furaltadone and artificial antigen, which is applied in the field of immunochemistry, can solve the problem that the AMOZ hapten cannot completely retain the characteristic structure of the competitor, and achieve the effects of enhanced immunogenicity, enhanced structural characteristics, and high sensitivity

Inactive Publication Date: 2017-04-26
广州润坤生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to overcome the traditional AMOZ hapten that cannot completely retain the characteristic structure of the competitor, and provide a preparation method and application of the furaltadone metabolite AMOZ derivatized hapten and artificial antigen, which can retain the competitive The hapten at the nitro end of the substance, artificial antigen, improves the specificity of the competitor

Method used

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  • Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen
  • Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen
  • Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Such as Figure 1-Figure 5 As shown, the furaltadone metabolite AMOZ derivatized hapten in Example 1 is prepared by the following method, and the steps are as follows:

[0052] (a') Dissolve 2.0 g (ie 7.1 mmol) of compound I in 10 ml of ethanol, adjust the pH value of the ethanol solution of compound I to 10-12 by adding 6 mol / L lithium hydroxide solution, and react at room temperature for 15 ~26h, then add 40ml of purified water, adjust the pH value of the resulting solution to 4~5 with 1M dilute hydrochloric acid, filter, and dry to obtain 1.1g of compound II.

[0053] ESI-MS: 167[M-CH2CH2CH2COOH-1], 252[M-1], 288[M+2H2O-1], 315[2×167-H2O-1], 505[2M-1], 537[2M +MeOH-1];

[0054] 1H NMR (600MHz, CDCl 3 , TMS): δ10.50(s, 1H), 8.20(d, 1H), 7.35(d, 1H), 7.15-7.19(dd, 1H), 4.23(t, 2H), 2.66(t, 2H), 2.26 (m, 2H).

[0055] (b') Dissolve 0.5g (ie 2.0mmol) of compound II in 10ml of methanol, add 0.48g (ie 2.4mmol) of furaltadone metabolite AMOZ, react at 60-70°C for 2h, a...

Embodiment 2

[0059] Example 2 is the artificial antigen of the furaltadone metabolite AMOZ. The artificial antigen includes an immunogen and a coating. The difference between the two lies in the types of carrier proteins coupled during the preparation process. For the hapten prepared in Example 1, the carrier protein is bovine serum albumin (BSA); the coating source is the hapten prepared in Example 1, and the carrier protein is ovalbumin (OVA).

[0060] The preparation method of the coating source of the furaltadone metabolite AMOZ of the present embodiment 2, the steps are as follows:

[0061] (1) Take 10.0 mg of the hapten of Example 1, dissolve it in 0.5 ml of dimethylformamide (DMF), stir well, add 8.0 mg of carbodiimide (EDC) and 6.0 mg of N-hydroxysuccinimide Amine (NHS), stirred at room temperature for 4-8h, to obtain hapten activated ester;

[0062] (2) Weigh 30.6mg of ovalbumin (OVA), make it fully dissolved in 4ml of PBS solution with a concentration of 0.01mol / L to form a carr...

Embodiment 3

[0074] The monoclonal antibody of the furaltadone metabolite AMOZ in Example 3 was prepared by the following method, the steps are as follows:

[0075] After emulsifying the immunogenic formula (III)-BSA of Example 2 with an equal volume of Freund's adjuvant, immunize BALB / C mice. The immunization dose for each mouse was 50-100 μg, and the immunization interval was 3 weeks. After 3 times of immunization, the blood of the tail vein of the mice was collected to detect the serum titer. If the antibody titer does not reach 60,000, booster immunization is required. After the antibody titer no longer rises, subcutaneous booster immunization is performed with 100 μg of immunogen, and the splenocytes of the mouse are fused with SP20 cells 5 days later. The fused cells were selected in HAT medium, and after 5 days, the complete medium was replaced with HAT medium for culture. Use ELISA to detect the cell supernatant, and carry out the limited dilution clone culture of the cells in the...

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Abstract

The invention discloses a furaltadone metabolite (AMOZ) derivatization hapten, a preparation method of an artificial antigen and an application of the artificial antigen. An active arm introduced by the hapten completely reserves feature structures of AMOZ derivatives; the electron cloud density is not influenced; active groups capable of being coupled to carrier protein are provided; the reaction condition is mild and the operation step is simple; and the prepared artificial antigen can be specifically combined with an antibody, has the characteristics of high potency, high sensitivity, high accuracy and the like, and can quickly detect residues of AMOZ in various products.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a preparation method and application of a furaltadone metabolite AMOZ derivatized hapten and an artificial antigen. Background technique [0002] Furans are a class of artificially synthesized antibiotics that share a 5-nitrofuran ring structure, and have antibacterial effects on Gram-positive bacteria, Gram-negative bacteria, and some fungi. Because of its low price, it is widely used in livestock, poultry and fish farming to treat enteritis and coccidiosis caused by Salmonella and Escherichia coli, and is often used as a growth promoter for livestock and aquatic fish and shrimp added to the feed. Nitrofurans mainly include furaltadone, nitrofurazone, nitrofurantoin and furazolidone. [0003] Among them, in terms of food safety, large or continuous use of furaltadone not only has toxic effects on farmed animals, but also has carcinogenic, teratogenic, and mutagenic eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D263/26C07K14/765C07K14/77C07K14/795C07K16/44G01N33/577
CPCC07D263/26C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/577
Inventor 胡睿
Owner 广州润坤生物科技有限公司
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