Olaquindox metabolin antigen, antibody, enzyme-linked immunosorbent assay kit and detection method

An enzyme-linked immunosorbent reagent and metabolite technology, which is applied in the field of drug residue detection, can solve problems such as the health of drug consumers and the threat of animal food exports.

Active Publication Date: 2019-01-11
广东标允生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, olaquindox is still widely illegally used or abused in production practice, and drug residues pose a huge potential threat to consumers' health and animal food exports, thus driving the research on related detection methods

Method used

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  • Olaquindox metabolin antigen, antibody, enzyme-linked immunosorbent assay kit and detection method
  • Olaquindox metabolin antigen, antibody, enzyme-linked immunosorbent assay kit and detection method
  • Olaquindox metabolin antigen, antibody, enzyme-linked immunosorbent assay kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Preparation of olaquindox metabolite hapten, immunogen, coating agent and monoclonal antibody

[0080] 1. Preparation of haptens, immunogens and coating agents

[0081] Using the active ester method, the hapten 4-(3-methyl-2-naphthylamino)butanoic acid (abbreviated as MNBA) was coupled to the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA), respectively, Prepare the immunogen MNBA-BSA and the coating original MNBA-OVA. The specific method is as follows:

[0082] (1) Hapten synthesis:

[0083] Dissolve 3-methyl-2-naphthoic acid (1.3 mmol) in SOCl 2 (11 mmol), microwave heated to 100°C for 2 h under nitrogen protection; the solvent was removed under reduced pressure, and the residue was dissolved in 1,4-dioxane (5 mL); under stirring conditions, the It was added dropwise until mixed with amino acid (1.3 mmol / L) and Na 2 CO 3 (3.2 mmol / L) in 5 mL aqueous solution; stir overnight at room temperature, add 20 mL hydrochloric acid solution (1 mo...

Embodiment 2

[0097] Example 2 Establishment of ELISA Method for Assaying Olaquindox Metabolites

[0098] (1) Optimizing the original coating concentration and antibody concentration, including the following steps:

[0099] 1) Dilute the coating agent with the coating solution according to the concentration of 15.6, 8.9, 5.0, 2.5, 1.25 ng / mL and coat the microtiter plate longitudinally, 100 μL / well, incubate overnight at 37°C, and wash twice with washing solution , pat dry on absorbent paper;

[0100] 2) Add 170 μL / well of the prepared blocking solution for blocking, overnight at 37°C, spin dry and put in an oven;

[0101] 3) Add 50 μL / well PBS solution diluted olaquindox metabolite standard solution;

[0102] 4) Add 50 μL / well of different gradients of olaquindox metabolite monoclonal antibodies (1:32000, 1:64000, 1:96000, 1:128000) diluted with PBS solution, incubate at 25°C for 30 min, and wash the plate 5 times , pat dry on absorbent paper;

[0103] 5) Add 100 μL / well of enzyme-labe...

Embodiment 3

[0116] Example 3 Preparation of Olaquindox Metabolite ELISA Kit

[0117] 1. Prepare reagents

[0118] (1) ELISA plate coated with olaquindox metabolite antigen

[0119] 96-well detachable ELISA plate, coated with olaquindox metabolite antigen and blocking solution, the coating concentration is 1.25 mg / L; the olaquindox metabolite is olaquindox metabolite hapten 4-(3-methyl- Conjugates of 2-naphthylamido)butyric acid and ovalbumin (OVA);

[0120] Coating of ELISA microplate: Dilute the coated antigen to 1.25 mg / L with coating solution, add 100 μL of coating solution to each well, incubate overnight at 37°C, pour off the liquid in the well, and wash twice with washing solution , and pat dry; then add 170 μL of blocking solution to each well, incubate at 37°C for 3 h, pour off the liquid in the well, dry in an oven at 37°C, and store in a vacuum-sealed aluminum foil bag at 4°C.

[0121] (2) Preparation of olaquindox metabolite standard solution

[0122] Accurately weigh the s...

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Abstract

The invention discloses an olaquindox metabolin antigen, an antibody, an enzyme-linked immunosorbent assay kit and a detection method. 4-(3-methyl-2-naphthalene carboxamide) butyrate is taken as an olaquindox metabolin hapten; the olaquindox metabolin antigen can be acquired after the hapten is coupled with carrier protein; the olaquindox metabolin antigen can be used for preparing an olaquindox metabolin specific antibody; the specific antibody can be used for preparing the enzyme-linked immunosorbent assay kit or colloidal gold test paper card used for detecting the residue of olaquindox metabolin in food. The invention also discloses the corresponding detection method of the kit. The method is simple, convenient and quick and is characterized by linear detection range within 0.2-16.2ng / mL, sensitivity of 0.54ng / mL, limit of detection of 0.32ng / mL, recovery rate within 80%-109.8%, low detection limit, high sensitivity, high specificity, high stability, low cost and suitability for mass sample detection and onsite quick detection.

Description

technical field [0001] The invention belongs to the technical field of drug residue detection. More specifically, it relates to a olaquindox metabolite antigen, antibody and ELISA detection kit and detection method. Background technique [0002] Olaquindox is an early product of quinoxaline drugs. Since the 1970s, it has been widely used all over the world, but it is currently banned from being used in food animals for growth promotion. The European Union banned the use of olaquindox and olaquindox in 1998, and my country banned the use of olaquindox and olaquindox for growth promotion of food animals, and stipulated the maximum residue limit of olaquindox residue markers in animal tissues. In recent years, olaquindox is mainly used in Australia, Brazil, Japan and China. [0003] The toxicity of olaquindox to animals mainly includes acute toxicity and cumulative toxicity. Excessive intake of olaquindox can cause stressful massive bleeding and death in animals, and when it...

Claims

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Application Information

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IPC IPC(8): C07C233/83C07C231/02C07K14/77C07K14/765C07K14/795C07K14/435C07K14/75C07K16/44G01N33/535G01N33/543G01N33/569
CPCC07C233/83C07K14/435C07K14/75C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/535G01N33/54306G01N33/56966
Inventor 杨金易沈玉栋曾道平谭庶孙远明陈丽韦田
Owner 广东标允生物科技有限公司
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