ELISA reagent for detecting malachite green and method

An enzyme-linked immunosorbent reagent, malachite green technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of time-consuming detection, expensive sample processing, complexity, etc., and achieve simple operation, simple sample pretreatment, and strong specificity Effect

Inactive Publication Date: 2009-05-06
深圳市绿诗源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two conventional methods for the detection of malachite green residues: HPLC and LC / MS, but they are relatively expensive and the sample processing is very complicated, and the detection is time-consuming

Method used

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  • ELISA reagent for detecting malachite green and method
  • ELISA reagent for detecting malachite green and method
  • ELISA reagent for detecting malachite green and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of ELISA Kit Components for Malachite Green Detection

[0048] 1. Antigen Synthesis

[0049] a. Synthesis of Coating Progen

[0050] The malachite green hapten is synthesized by the derivative method, and then the hapten is obtained by coupling the hapten with the bovine gamma globulin carrier protein by the active ester method through the diazotization reaction.

[0051] b. Synthesis of Immunogen

[0052] The malachite green hapten is obtained by coupling the malachite green hapten with the keyhole limpet hemocyanin carrier protein by the active ester method through diazotization reaction.

[0053] 2. Preparation of Malachite Green Mouse Monoclonal Antibody

[0054] a. Animal immunity

[0055] Balb / c mice were used as immunized animals, and the conjugated malachite green hapten and keyhole limpet hemocyanin was used as the immunogen, and the immunization dose was 300 μg / mouse. The adjuvant was mixed to make an emulsifier, which was injected s...

Embodiment 2

[0070] Example 2 Detecting the formation of malachite green monoclonal ELISA kit

[0071] Set up the enzyme-linked immunosorbent assay kit for detection of malachite green, so that it contains the following components:

[0072] (1) enzyme-linked plate coated with malachite green antigen;

[0073] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0074] (3) malachite green mouse monoclonal antibody;

[0075] (4) 6 bottles of malachite green standard solution, the concentrations are 0μg / L, 0.01μg / L, 0.05μg / L, 0.1μg / L, 0.5μg / L, 1μg / L;

[0076] (5) The substrate chromogenic solution is a mixed solution of hydrogen peroxide or carbamide peroxide and o-phenylenediamine (OPD) or tetramethylbenzidine (TMB);

[0077] (6) The stop solution is 2mol / L sulfuric acid buffer;

[0078] (7) Concentrated washing solution: phosphate buffer containing 0.1% to 0.5% Tween 80 and 0.5% sodium azide;

[0079] (8) Antibody diluent is pH 8.2, 0.05 mol / L, phosphate buffer cont...

Embodiment 3

[0081] Example 3 Detecting the formation of malachite green polyclonal ELISA kit

[0082] Set up the enzyme-linked immunosorbent assay kit for detection of malachite green, so that it contains the following components:

[0083] (1) Enzyme-linked plate coated with goat anti-rabbit anti-antibody;

[0084] (2) malachite green antigen labeled with alkaline phosphatase;

[0085] (3) Malachite green rabbit polyclonal antibody;

[0086] (4) 6 bottles of malachite green standard solution, the concentrations are 0μg / L, 0.01μg / L, 0.05μg / L, 0.1μg / L, 0.5μg / L, 1μg / L;

[0087] (5) Substrate chromogenic solution p-nitrophosphate buffer;

[0088] (6) The stop solution is 2mol / L sodium hydroxide buffer solution;

[0089] (7) Concentrated washing solution containing 0.1% to 0.5% Tween 80, 0.5% sodium azide in phosphate buffer, 1 bottle;

[0090] (8) The concentrated complex solution is a phosphate buffer solution containing 20% ​​methanol and 1% bovine serum (BSA).

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Abstract

The invention discloses an Elisa agent for detecting malachite green and a method thereof. The Elisa agent comprises a malachite green antigen or antibody coated Elisa plate, an enzyme label, a malachite green specific antibody, a malachite green standard solution, a substrate developing solution, a stop solution, a concentrated cleaning solution, enzyme label diluents and a concentrated combined solution. The Elisa agent is used for the quantitative detection of the content of the malachite green in fishes, shrimps and water samples and has the advantages of high specificity and sensitivity, easy sample pretreatment, short detection time, large sample detection amount, and the like.

Description

technical field [0001] The invention relates to an ELISA reagent and method for detecting malachite green in the technical field of ELISA and veterinary drug residue detection and analysis. Background technique [0002] Malachite Green (MG) is widely used in fish farming as an antifungal, anti-ectoparasitic and disinfectant. However, recent studies have shown that malachite green is highly carcinogenic. Therefore, many countries have listed malachite green as a banned drug for aquaculture. In May 2002, my country also included malachite green in the "List of Veterinary Drugs and Their Compounds Prohibited for Food Animals", prohibiting them from being used in all food animals. However, malachite green is still banned in aquaculture because of its low price and remarkable fungicidal effect. [0003] In order to prevent malachite green residues from entering the food chain, farmers and government monitoring departments need a fast, accurate and sensitive technology to detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
Inventor 聂继斌唐俊谭彩莲杨宏温俊梅齐欣李成杨宗繁
Owner 深圳市绿诗源生物技术有限公司
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