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Preparation method and application of derived hapten and artificial antigen of furazolidone metabolite AOZ

A furazolidone and artificial antigen technology, applied in the field of immunochemistry, can solve the problem of inability to completely retain the characteristic structure of competitors, and achieve the effects of enhanced immunogenicity, high sensitivity and strong specificity

Inactive Publication Date: 2017-06-20
广州润坤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to overcome the traditional AOZ hapten that cannot completely retain the characteristic structure of the competitor, and to provide a preparation method and application of the furazolidone metabolite AOZ derivatized hapten and artificial antigen, which can retain the competitor nitrate Base-terminal haptens, artificial antigens, improve competitor specificity

Method used

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  • Preparation method and application of derived hapten and artificial antigen of furazolidone metabolite AOZ
  • Preparation method and application of derived hapten and artificial antigen of furazolidone metabolite AOZ
  • Preparation method and application of derived hapten and artificial antigen of furazolidone metabolite AOZ

Examples

Experimental program
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Embodiment 1

[0049] Such as Figure 1-Figure 5 As shown, the furazolidone metabolite AOZ derivatized hapten in Example 1 is prepared by the following method, and the steps are as follows:

[0050] (a) Dissolve 2.0 g (7.1 mmol) of compound I in 10 ml of ethanol, add 6 mol / L lithium hydroxide solution to adjust the pH value of the ethanol solution of compound I to 10-12, and react at room temperature for 15 to 26 hours, Then 40ml of purified water was added, and the resulting solution was passed through 1M dilute hydrochloric acid to adjust the pH value to 4-5, filtered, and dried to obtain 1.1g of compound II.

[0051] 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];

[0052] 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).

[0053] (b) Dissolve 0.5g (ie 2.0mmol) of compound II in 10ml of methanol, add 0.24g (ie 2.4mmol) of furazolidone metabolite AOZ,...

Embodiment 2

[0057] Example 2 is an artificial antigen of furazolidone metabolite AOZ, and the artificial antigen includes an immunogen and a coating agent. The difference between the immunogen and the coating source is the type of carrier protein that is coupled, the immunogen uses the hapten of Example 1, and the carrier protein uses bovine serum albumin (BSA); The hapten and the carrier protein are ovalbumin (OVA).

[0058] In Example 2, the coating source of the furazolidone metabolite AOZ was prepared by the following method, and the steps were as follows:

[0059] (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 EDC and 6.0 mg of N-hydroxysuccinimide (NHS), and After stirring for 4 hours, the hapten-activated ester can be obtained;

[0060] (2) Take 30.6mg ovalbumin (OVA), make it fully dissolved in 4ml of PBS solution with a concentration of 0.01mol / L to form a carrier protein solution, and slowly dissolve the ...

Embodiment 3

[0072] The monoclonal antibody of the furazolidone metabolite of this embodiment is prepared by the following method, and the steps are as follows:

[0073] After emulsifying the immunogenic formula (Ⅲ)-BSA of Implementation 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. After 5 days, mouse splenocytes are taken to fuse with SP20 cells. 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...

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Abstract

The invention discloses a preparation method and application of a derived hapten and an artificial antigen of a furazolidone metabolite AOZ. An active arm introduced into the hapten completely retains a feature structure of an AOZ derivative and has no influence on electron cloud density of the AOZ derivative; in addition, the active arm has an active group which can be coupled with carrier protein and is mild in reaction conditions and simple in operation step; the prepared artificial antigen can be specifically combined with an antibody, has the characteristics of high titer, high sensitivity, high accuracy and the like, and can be used for quickly detecting residues of the furazolidone metabolites 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 furazolidone metabolite AOZ 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. However, in terms of food safety, large or continuous use of furazolidone not only has toxic effects on farmed animals, but also has carcinogenic, teratogenic, and mutagenic effects, and i...

Claims

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Application Information

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IPC IPC(8): C07D263/26C07K14/765C07K14/77C07K14/795C07K16/44G01N33/531
CPCC07D263/26C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/531
Inventor 胡睿李斌
Owner 广州润坤生物科技有限公司
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