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Enzyme-linked immunosorbent assay kit for detecting melamine and application thereof

An enzyme-linked immunosorbent reagent, melamine technology, applied in the direction of testing food, testing eggs, testing dairy products, etc., can solve the problems of cumbersome process, unsuitable for on-site monitoring and screening of a large number of samples, complex instruments and equipment, etc.

Inactive Publication Date: 2012-11-14
河北滦牧农业开发股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current chemical methods for detecting melamine content include high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Due to the complicated equipment and cumbersome process, they are not suitable for on-site monitoring and screening of a large number of samples.

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting melamine and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting melamine and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting melamine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, melamine hapten, melamine antigen, melamine monoclonal antibody and preparation thereof

[0065] 1. Synthesis and identification of melamine hapten

[0066] 1. Synthesis of melamine hapten

[0067] 1) Weigh 120 mg of 4,6-diamino-2-hydroxy-1,3,5-triazine (purchased from Acros Organics, CAS No.: 3397-62-4), 100 μL of ethyl 4-bromobutyrate, Put 100 mg of anhydrous sodium carbonate in a 50 mL flask, add 20 mL of acetone, reflux at 70°C for 16 hours, and evaporate the solvent to dryness under reduced pressure.

[0068] 2) The residue was dissolved in 15 mL methanol-potassium hydroxide mixture (15%, 88 mg potassium hydroxide dissolved in 5 mL methanol), refluxed at 80° C. for 1 hour, and the solvent was evaporated to dryness under reduced pressure.

[0069] 3) Add 8 mL of distilled water to the residue (all dissolved), acidify with 2M HCl to pH 3 (white precipitate appears).

[0070] 4) Collect the precipitate by centrifugation and dry at 50°C. 143 mg of the...

Embodiment 2

[0107] Embodiment 2, the ELISA kit that detects melamine

[0108] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0109] 1. Melamine coating agent; as described in Example 1.

[0110] 2. ELISA plate;

[0111] 3. Melamine monoclonal antibody: secreted by the melamine monoclonal antibody hybridoma cell line MAL with the preservation number CGMCC No.3394;

[0112] 4. Melamine standard: the concentration of the standard solution is 0 μg / L, 5 μg / L, 20 μg / L, 100 μg / L, 500 μg / L and 2500 μg / L; the melamine standard is purchased from Sigma Aldrich; CAS number: 108- 78-1; Dilute to the above concentrations with sample diluent;

[0113] 5. Goat anti-mouse secondary antibody labeled with horseradish peroxidase;

[0114] 6. Substrate chromogenic solution: composed of liquid A and liquid B, liquid A is an aqueous solution of 2% carbamide peroxide, liquid B is an aqueous solution of 1% tetramethylbenzidine;

[0115] 7. Termination solution: 2mol / L aqu...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting melamine and application thereof. The enzyme-linked immunosorbent assay kit comprises melamine monoclonal antibody and melamine hapten. The melamine monoclonal antibody is secreted by a melamine monoclonal antibody hybridoma cell strain MAL with the collection number of CGMCC No.3394. The enzyme-linked immunosorbent assay kit mainly performs qualitative or quantitative detection on the content of melamine in edible products for animals and human (particularly milk products such as milk and milk powder, eggs, feeds and the like) by an indirect competitive ELISA method. The kit and the detection method have low requirement on pretreatment of a sample; the pretreatment process of the sample is simple; and massive samples can be simultaneously detected quickly. The melamine monoclonal antibody with high specificity is adopted, and the detection method is convenient and practicable and has the characteristics ofhigh specificity, high sensitivity, high accuracy and the like.

Description

technical field [0001] The invention relates to an ELISA kit for detecting melamine and its application. Background technique [0002] Melamine, molecular formula C 3 N 6 h 6 , referred to as triamine, is a triazine nitrogen-containing heterocyclic organic compound. Melamine is weakly alkaline and can form melamine salts with hydrochloric acid, sulfuric acid, nitric acid, acetic acid, oxalic acid, etc. Melamine is an important nitrogen-heterocyclic organic chemical raw material. In neutral or slightly alkaline conditions, it can condense with formaldehyde to form various methylol melamines, but in slightly acidic environments (pH5.5-6.5) The derivatives undergo polycondensation reaction to form resin products. Melamine is a basic organic chemical intermediate product with a wide range of uses, and its main use is as a raw material for the production of melamine-formaldehyde resin. The resin has higher hardness than urea-formaldehyde resin, non-flammable, water resistan...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/04G01N33/08G01N33/10G01N33/02
Inventor 吴小平江海洋王战辉史为民徐飞李娜王熙米铁军王进
Owner 河北滦牧农业开发股份有限公司
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