ELISA kit capable of rapid detection of diminazene aceturate residues in animal-derived food

An enzyme-linked immunosorbent reagent and technology of triazine amidine, which is applied in the field of specific monoclonal antibody, preparation of monoclonal antibody and its enzyme-linked immunosorbent detection kit, and enzyme-linked immunological detection, can solve the problem of small safety range, slow elimination, animal It is prone to adverse reactions and other problems, achieving the effect of reducing operation steps, saving time, and identifying a wide range of spectra

Inactive Publication Date: 2017-02-15
ZHENJIANG XIANCHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the existing clinical reports, the safety range of this drug is small, and when it is used continuously or in large doses, animals are prone to adverse reactions. After entering the animal body, triazamidine absorbs and distributes rapidly, and eliminates slowly, and is mainly excreted through the kidneys in its original form. In vitro, virtually no metabolism in vivo

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of kit components

[0043] 1. Antigen synthesis

[0044] a. Dissolve 10 g of carrier bovine serum albumin (BSA) in 35 mL of pH9.6 carbonate buffer, add 10 g of ethylenediamine for activation;

[0045] b. Dissolve 1 g of triazamidine in 10 ml of 0.5 M sodium hydroxide solution;

[0046] c. Dissolve 1 g of carbodiimide in 5 ml of pure water, then add it to the amidine triazine solution and stir at room temperature for 3 hours;

[0047] d. Add the activated carrier protein BSA dropwise to the triazine solution and stir at 4°C overnight;

[0048] e. The artificial antigen obtained after the reaction was dialyzed against 0.1 M PBS buffer for 5 days, and the buffer was changed 4 times a day; the obtained purified artificial antigen was concentrated by ultrafiltration or lyophilized for storage.

[0049] 2. Preparation of triazamidine monoclonal antibody

[0050]a. Animal immunization procedure: Use triazine artificial antigen as the immunogen to...

Embodiment 2

[0065] Example 2 Establishment of an enzyme-linked immunosorbent assay kit for the rapid detection of triazine residues in food of animal origin

[0066] This ELISA kit consists of the following components:

[0067] (1) Enzyme plate coated with triazine;

[0068] (2) Triazine monoclonal antibody working solution with a protein concentration of 0.5 ug / L;

[0069] (3) Triazine standard with a concentration of 1 μg / mL;

[0070] (4) Standard diluent: 0.05 mol / L Tris-HCl, pH 8.0, 0.9% NaCl buffer;

[0071] (5) Goat anti-mouse enzyme-labeled secondary antibody working solution labeled with horseradish peroxidase;

[0072] (6) Hydrogen peroxide at the liquid level of substrate chromogenic solution A, and o-phenylenediamine at the liquid level of substrate chromogenic solution B;

[0073] (7) The washing buffer is 0.05 mol / L Tris-HCl, pH 8.0, 0.9% NaCl, 0.04% Tween20.

[0074] (8) The stop solution is 2 mol / L sulfuric acid.

Embodiment 3

[0075] Example 3 Detection of triazine residues in samples

[0076] 1. Sample pretreatment

[0077] a. Dairy products: Skim milk can be directly measured with PBS buffer according to the skim milk:PBS ratio of 1:10, and whole milk needs to be centrifuged to remove fat, and then diluted according to the ratio of 1:10 for measurement;

[0078] b. Tissue samples: Homogenize lean meat or visceral tissue samples with a homogenizer, accurately weigh 4 g of samples, add 4 mL of ethyl acetate, shake for 10 minutes, centrifuge at room temperature at 4000 rpm / min for 15 minutes, and take out 2 mL of the upper layer , dry at 50-60°C under nitrogen flow, add 1 mL of n-hexane to dissolve the dried residue, add 1 mL of standard diluent, shake vigorously for 1 min, centrifuge at room temperature 4000 rpm / min for 5 minutes, remove the upper oil phase, and take the lower layer 50 μl of the aqueous phase was used for determination;

[0079] c. Urine: Take 2 mL of urine into a centrifuge ...

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PUM

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Abstract

The invention provides an ELISA kit capable of rapid detection of diminazene aceturate residues in animal-derived food, belonging to the field of immunoassay and detection technology. The kit comprises an enzyme-labeled plate coated with a diminazene aceturate antigen, a diminazene aceturate antibody, a standard diminazene aceturate substance, a diluted standard substance solution, an enzyme-labeled second antibody, a substrate developing solution, a washing buffer and a stopping solution. The kit provided by the invention employs a highly specific monoclonal antibody against diminazene aceturate, and main reagents are provided in the form of a working solution, so operation steps are reduced and time is saved. Meanwhile, since the monoclonal antibody against diminazene aceturate has a wide identification spectrum, the kit is capable of detecting the total amount of diminazene aceturate residues in animal-derived food, especially in meat products, aquatic products, bee products and dairy products.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunoassay detection, and in particular relates to the preparation of a monoclonal antibody capable of recognizing triazine drugs and an enzyme-linked immunoassay kit. The monoclonal antibody of the present invention is secreted by the hybridoma cell line C2F3C2 established by the applicant. The invention discloses a preparation method of a specific monoclonal antibody, a coating agent and an immunogen and an enzyme-linked immunoassay method. Compared with the prior art, the kit and method of the present invention have the advantages of simplicity, rapidity, sensitivity, accuracy, etc., and can simultaneously detect triazine in food of animal origin, especially in meat products, aquatic products, bee products and dairy products The total amount of drug residues. Background technique [0002] Triazamidine, also known as Xuechongjing and Bernier, is a yellow or golden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/02
CPCG01N33/02G01N33/577
Inventor 洪霞刘静
Owner ZHENJIANG XIANCHUANG BIOTECH CO LTD
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