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Immune testing method for enzyme linked retained 3-methylquinoxaline-2-carboxylic acid and reagent set

An enzyme-linked immunoassay and methylquinoxoline technology, which is applied in the field of immunochemical analysis, can solve the problems of no MQCA residue enzyme-linked immunosorbent analysis technology, etc., and achieve reliable detection results, high sensitivity, and high accuracy.

Inactive Publication Date: 2007-05-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no patents and literature reports on MQCA residue enzyme-linked immunosorbent assay technology at home and abroad

Method used

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  • Immune testing method for enzyme linked retained 3-methylquinoxaline-2-carboxylic acid and reagent set

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Embodiment 1

[0030] Embodiment 1, the preparation of antigen

[0031] The preparation of the antigen of the present invention includes the preparation of the immunogen and the coating original, and its specific synthesis route is shown in FIG. 1 .

[0032] 1.1 Preparation of immunogen

[0033] Get 0.0376g of 3-methylquinoxaline-2-carboxylic acid (MQCA), 0.0116g of N-hydroxysuccinimide (NHS), and 0.0434g of N, N-dicyclohexylcarbodiimide (DCC) into Stir in 1.5mL 1,4-dioxane overnight at room temperature; centrifuge to remove the precipitate, then mix with 5mL bovine serum albumin (BSA) 0.34g solution and stir overnight at 4°C; centrifuge, supernatant with PBS0.1mol / L, pH7.4 phosphate buffer solution, dialyzed for 3 days, freeze-dried, and stored at 4°C for later use. The ultraviolet spectrum of the obtained immunogen is shown in FIG. 2 .

[0034] 1.2 Preparation of coating agent

[0035] Get 0.0376g of 3-methylquinoxaline-2-carboxylic acid (MQCA), 0.0116g of N-hydroxysuccinimide (NHS), ...

Embodiment 2

[0036] Embodiment 2, the preparation of antibody

[0037] 2.1 Immunization of animals

[0038] New Zealand white rabbits were used as immunized animals, MQCA and BSA conjugates were used as immunogens, and the immunization dose was 0.5-1 mg / rat. For the first immunization, the immunogen was mixed with the same amount of Freund's complete adjuvant to make an emulsifier. New Zealand white rabbits were injected subcutaneously at multiple points on the back of the neck, taking the same dose of immunogen plus the same amount of Freund's incomplete adjuvant mixed and emulsified at intervals of 2 to 4 weeks, and boosted the immunization once. During the immunization period, the serum antibody titer and specificity should be monitored, and the last time Without adjuvant. Blood was collected 7-10 days after the last immunization, blood was exsanguinated from the carotid artery, and the purified MQCA polyclonal antibody was obtained by fractional precipitation with ammonium sulfate.

...

Embodiment 3

[0052] Embodiment 3, establishment of sample pretreatment method

[0053]Extraction: Accurately weigh 5 g of porcine muscle tissue and place it in a centrifuge tube with stopper, add 8 mL of 5% metaphosphoric acid and 20% methanol solution, vortex for 2 min, centrifuge at 6000 r / min at 25 °C for 15 min, take out the supernatant, and then Add 8 mL of 5% metaphosphoric acid and 20% methanol solution to the tissue sample and repeat the extraction once, and combine the two supernatants. Add 8 mL of ethyl acetate to the supernatant, vortex for 1 min, centrifuge at 4000 r / min for 10 min, take the organic layer, add 8 mL of ethyl acetate to repeat the extraction, and combine the organic phases twice. Add 6 mL of phosphate buffer to the organic phase, vortex for 1 min, and let stand for 10 min to make the lower layer clear, then collect the aqueous phase. Another 6 mL of phosphate buffer was used to extract the ethyl acetate phase once. The aqueous phase was cleared and the 2 aqueou...

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Abstract

This invention discloses one enzyme immune test method and agent case for 3- methyl quinoline 2-carboxyl acid in immune chemical analysis technique field, which comprises the following steps: processing immune antigen and cover antigen and alloantibody and pre-processing sample and establishing ELISA method; the matched agent case is composed of 3- methyl quinoline 2-carboxyl acid alloantibody covered by MQCA and egg albumin couple and MQCA standard sample; the sample is processed through metaphosphoric acid to release MQCA and MAX and indirectly competition ELISA method.

Description

technical field [0001] The invention belongs to the technical field of immunochemical analysis, and in particular relates to an enzyme-linked immunoassay method and kit for 3-methylquinoxaline-2-carboxylic acid (abbreviated as MQCA, the same below) residue, which is suitable for determining animal sex Residues of MQCA, the indicator substance of olaquindox residues in food. Background technique [0002] Lalaquindox, also known as quinamide alcohol, quinodos, and beyonol, belongs to quinoxaline-1,4-dioxide drugs and is a synthetic broad-spectrum antibacterial drug. It was once widely used as a growth-promoting agent in livestock , poultry, and aquaculture. Toxicological studies have found that olaquindox is carcinogenic and mutagenic. Olaquindox is quickly metabolized in animals, and MQCA finally leaves the body and is regarded as a residual marker of olaquindox. Therefore, the analysis of olaquindox residues usually analyzes its residue marker MQCA. The maximum residue l...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/547
Inventor 袁宗辉杨波赵春保高爱中彭大鹏王玉莲谢长清陶燕飞陈冬梅黄玲利戴梦红刘振利
Owner HUAZHONG AGRI UNIV
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