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Monoclonal antibody of leucomalachite green and preparation method and usage thereof

A colorless malachite green and monoclonal antibody technology, applied in the field of biological immunology, can solve the problems of high cost, high cost, and long time spent, and achieve high sensitivity, good stability, and strong specificity

Inactive Publication Date: 2013-09-18
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although thin-layer chromatography (TLC), spectrophotometry, and high-performance liquid chromatography (HPLC) have high sensitivity and can perform large-scale detection, they take a long time to detect and the cost of purchasing instruments is also very high. Generally, each The detector is more than 300,000 yuan, and the cost is extremely high

Method used

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  • Monoclonal antibody of leucomalachite green and preparation method and usage thereof
  • Monoclonal antibody of leucomalachite green and preparation method and usage thereof
  • Monoclonal antibody of leucomalachite green and preparation method and usage thereof

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Experimental program
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Effect test

Embodiment 1

[0033] 1. Amino colorless malachite green (NH 2 -LMG) synthesis

[0034] 1. Weigh 0.2g of colorless malachite green and dissolve it in 2mL of concentrated sulfuric acid, stir and heat to maintain the temperature at 90°C, add dropwise a mixture of 2mL of concentrated sulfuric acid and 2mL of concentrated nitric acid, react for 2 hours, and add the reaction solution to 3-10mL of ice water after cooling , drop sodium bicarbonate to neutralize to alkaline (pH 7.5-10), yellow crystals precipitate out;

[0035] 2. Add 10mL of hydrochloric acid solution to the obtained yellow crystals, then add 1g of zinc powder, react for 5 hours at 85-95°C oil bath temperature, add 15mL of distilled water after the reaction is completely cooled, stir and filter to remove solid impurities, and the filtrate is oxidized with hydrogen After the sodium is neutralized to alkaline (pH 7.5-10), a large amount of precipitation appears immediately, and the residue is vacuum-dried to obtain a purple solid, w...

Embodiment 2

[0084] In order to verify the effectiveness of the monoclonal antibody secreted by the hybridoma cell line obtained in the present invention, the following assays were carried out.

[0085] 1. Determination of monoclonal antibody affinity constant

[0086]The affinity of the monoclonal antibodies was determined using a non-competitive enzyme immunoassay. Coat the original LMG-OVA with 200, 100, 50, 25 ng / ml to the ELISA plate, 100 μl / well, 37°C, 1 hour; use PBS containing 5% milk to block the monoclonal antibody from 1000 times Begin with doubling dilution, and the rest are the same as the indirect ELISA detection method. Taking the logarithmic value of the antibody dilution factor as the abscissa and its corresponding A value as the ordinate, four S-shaped curves can be drawn in one coordinate system. Find the top of the S-curve and set it as Amax. Find the antibody concentration corresponding to 50% Amax of each of the four curves in the curves. Two groups of 4 concentra...

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Abstract

The invention relates to a monoclonal antibody of leucomalachite green, a hybridoma cell strain producing the monoclonal antibody and a preparation method and usage of the monoclonal antibody. The invention also relates to a composition containing the monoclonal antibody and a detection kit. According to the invention, the monoclonal antibody of leucomalachite green is successfully prepared, the monoclonal antibody has high sensitivity and strong specificity and only has cross reaction with malachite green while having no cross reaction with parafuchsin, methyl blue, crystal violet, ciprofloxacin and chloramphenicol, thereby providing key technology for rapid detection of residual leucomalachite green aquatic products and laying a foundation for research and development of the detection kit of the leucomalachite green.

Description

technical field [0001] The invention relates to the technical field of biological immunology, in particular to a colorless malachite green monoclonal antibody and a preparation method and use thereof. Background technique [0002] The chemical name of malachite green (Malachite Green, MG) is tetramethyldiaminotriphenylmethane, and the molecular formula is C 23 h 25 ClN 2 , also known as basic green, malachite green or China green, is a triphenylmethane dye, which has been widely used in the treatment and prevention of saprolegniasis. However, malachite green and its metabolite leuco malachite green (Leuco malachite green, LMG) have side effects such as carcinogenic, teratogenic, mutagenic, etc., especially leuco malachite green, which is more common in aquatic products than malachite green There are more residues and a longer residue time. The country has explicitly banned its use in the treatment and prevention of aquatic animal diseases, but there are still many unscrup...

Claims

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Application Information

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IPC IPC(8): C07K16/00C12P21/08C12N5/20G01N33/577C12R1/91
Inventor 姜有声胡鲲陈力杨先乐喻文娟
Owner SHANGHAI OCEAN UNIV
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