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Kit for detecting nifurthiazole metabolite and its use method

A technology of nifursol and metabolites, which is applied in the field of immunochemical analysis, can solve the problems of being unable to cope with the rapid detection of large quantities of samples in a short time, and achieve high stability and sensitivity, good specificity, and fast operation

Inactive Publication Date: 2009-04-29
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrument method is obviously unable to cope with the rapid detection of a large number of samples in a short time

Method used

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  • Kit for detecting nifurthiazole metabolite and its use method
  • Kit for detecting nifurthiazole metabolite and its use method
  • Kit for detecting nifurthiazole metabolite and its use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Synthesis of Haptens

[0025] Dissolve 0.11 g of glyoxylic acid in 10 mL of methanol, add 0.24 g of 3,5-dinitrosalicylic acid hydrazine (DNSH), stir for 3 h, filter and wash the precipitate with methanol to obtain 0.21 g of the hapten DNSHA, yield 72% . APCI-MS m / z: 297[M-H] - .APCI-MS / MS m / z: 281 [M-OH] - ; 281(M-OH); 226(M-NCHCOOH); 210(M-H + -NHNCHCOOH); 183(M-H + -2CONHNCHCOOH). 1 H NMR (600MHz, d 6 -DMSO, TMS): δ 14.13(s, 1H); 8.78(d, J=3.0Hz, 1H); 8.580(d, J=3.0Hz, 1H); 7.95(s, 1H); 7.70(s, 1H ).

Embodiment 2

[0026] Example 2 Synthesis of Antigen

[0027] Dissolve 0.1mmol of the hapten DNSHA in 2mL of DMF, stir and add DCC27.5mg and NHS14.4mg, and react overnight with magnetic stirring at 4°C. After centrifugation, the supernatant is liquid A; Add 1 mL of DMF to PBS (PH8.0), stir and dissolve to prepare solution B; under magnetic stirring, gradually drop solution A into solution B, and react at 4°C for 12 hours. After centrifugation, take the supernatant and dialyze with normal saline at 4°C for 3 days, and change the dialysate 3 times a day. The whole antigen DNSHA-BSA obtained is aliquoted at a concentration of 1g / L and stored in a -20°C refrigerator for immunization . The structural formula of the prepared whole antigen DNSHA-BSA is:

[0028]

Embodiment 3

[0029] Example 3 Synthesis of Coating Progen

[0030] Dissolve 0.1mmol of 3,5-dinitrosalicylic acid (DNSA) in 2mL of DMF, stir and add 27.5mg of DCC and 14.4mg of NHS, and react overnight with magnetic stirring at 4°C. After centrifugation, the supernatant is liquid A; weigh 140mg of BSA Dissolve in 10 mL of PBS (pH 8.0) with a concentration of 0.1 mol / L, add 1 mL of DMF, stir and dissolve to prepare liquid B; under magnetic stirring, liquid A is gradually dropped into liquid B, and react at 4°C for 12 hours. After centrifugation, take the supernatant night, dialyze with normal saline at 4°C for 3 days, change the dialysate 3 times a day, and pack the obtained coated original DNSA-OVA at a concentration of 1g / L, and freeze it in a -20°C refrigerator for packaging. be used.

[0031] The prepared coating former DNSA-OVA structural formula is:

[0032]

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Abstract

The invention discloses a reagent kit for detecting a nifursol metabolin. The reagent kit comprises a kit body, a specific antibody solution, a 3,5-dinitro salicylic hydrazine (DNSH) standard solution, an enzyme label plate, a sample diluent, a washing solution, a second antibody solution, a chromogenic A solution, a chromogenic B solution and a stopping solution. A sample to be detected is hydrolyzed through hydrochloric acid, is degreased through aether; protein is removed through methanol; the sample is redissolved through the sample diluent and is subjected to other sample pretreatment steps; and the sample is analyzed by using the reagent kit. The reagent kit is suitable for detecting the nifursol metabolin and has the advantages of simple and rapid pretreatment, high flux amount, low price and the like; and the lowest detection limit is 0.1ng / mL.

Description

technical field [0001] The invention belongs to the field of immunochemical analysis, and in particular relates to an enzyme-linked immunoimmunoassay kit for analyzing and detecting nifursol metabolites and a use method thereof. Background technique [0002] Nifursol is a nitrofuran drug, it has a very good antibacterial effect, in addition to being used as an antibiotic in veterinary medicine, it was also widely used as a growth promoter for pigs, poultry and aquatic products. But because of its possible unsafe factors, the EU legislated in 2002 to ban its use in animal-derived food. Studies have proved that nifursol is sensitive to light and metabolized quickly. It can be metabolized rapidly in animals within a few hours. The metabolite is 3,5-dinitrosalicylic acid hydrazine (DNSH), which can stay in the body for a long time. . Therefore, the main target of analysis of nifursol is its metabolite. [0003] For the determination of DNSH residues in animal-derived foods, t...

Claims

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Application Information

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IPC IPC(8): G01N33/543
Inventor 沈玉栋张世伟孙远明王弘雷红涛肖治理杨金易
Owner SOUTH CHINA AGRI UNIV