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Indirectly racing ELISA kit for detecting leuco malachite green

A technology of recessive malachite green and enzyme-linked immunosorbent reagents, which is applied in the field of enzyme-linked immunosorbent immunoassay and veterinary drug residue detection and food safety, can solve the problems of high cost of liquid chromatography, inability to realize on-site detection, long operation time, etc., and achieve Simple operation steps, no radioactive isotope contamination, and high degree of automation

Active Publication Date: 2009-06-10
WUXI ZODOLABS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance liquid chromatography is costly, takes a long time to operate, and has many and complicated steps, so it cannot achieve real on-site detection

Method used

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  • Indirectly racing ELISA kit for detecting leuco malachite green
  • Indirectly racing ELISA kit for detecting leuco malachite green
  • Indirectly racing ELISA kit for detecting leuco malachite green

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Hapten and complete antigen (immunogen) synthesis

[0024] 1.1 Reagents and instruments

[0025] Recessive malachite green (Beijing Chemical Reagent Co., Ltd.), bovine serum albumin (BSA), keyhole limpet ceruloplasmin (KLH), etc. (purchased from Beijing Dingguo Biotechnology Co., Ltd.), all reagents used are chemically pure or analytical pure.

[0026] Yanco micro melting point apparatus (the thermometer was not calibrated); Bruker AMX-300 nuclear magnetic resonance apparatus (IMS was the internal standard, CDCl 3 as a solvent). Double-beam UV-Vis spectrophotometer (TM-1909, Beijing Puxi General Instrument Co., Ltd.), magnetic stirrer (Shanghai Dongrongfeng Scientific Instrument Co., Ltd.), desktop centrifuge (Minispin maximum speed 13400rpm, maximum centrifugal force 12100rcf, 2mL× 12), ZS-2 automatic plate microplate reader (Beijing Xinfeng Electric Technology Co., Ltd.), water-proof electric heating constant temperature incubator (Shanghai Yuejin Medical...

Embodiment 2

[0037] Example 2 Preparation and Titer Detection of Antibodies

[0038] 2.1 Antibody preparation

[0039] Preparation of polyclonal antibodies Select 3 healthy male New Zealand white rabbits weighing 2-2.5kg, use BSA-hapten as the immunogen and the same amount of Freund's complete adjuvant to mix them into a water-in-oil emulsion by syringe pumping method, and weigh 1mg The amount per kg body weight was used for the first immunization, and multi-point subcutaneous injections were taken on the back. Booster immunization every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the dose and method are the same as the first immunization. From the third immunization, 10 days after each immunization, 1 mL of blood was collected from the ear vein for antibody titer detection. When the antibody titer no longer increased, the last (7th) immunization was performed without adjuvant. Thigh intramuscular injection, carotid artery bloodletting after 7 days...

Embodiment 3

[0049] Example 3 Preparation of enzyme-labeled antigen

[0050] The preparation of the enzyme-labeled antigen by the active ester method is achieved as follows: take 10mol of the recessive malachite green hapten, 10μmol of N-hydroxysuccinimide (NHS) and 10μmol of cyclodihexylcarbodiimide (DCC), and use 1mL di Dissolve the mixture in methylformamide (DFM), react overnight in the dark at room temperature, centrifuge to remove the precipitate, take the supernatant to dry, add it to 10 mL of borate buffer containing 200 mg of horseradish peroxidase (HRP) (Ph9.0), the mixture was magnetically stirred at 4°C for 6 hours, dialyzed overnight at 4°C for 4 times (phosphate buffer at pH 7.4), and the results of full-wavelength scanning by a UV scanner inferred successful coupling .

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Abstract

The invention discloses an ELISA kit for detecting recessive malachite green, comprising recessive malachite green polyclonal antibody, recessive malachite green hapten and complete antigen and enzyme-labelled antigen. The inventive ELISA kit is sensitive, fast and accurate, mainly used for screening of mass samples. Mainly reagent in the kit is provided in the form of work liquid, with the advantages of convenient usage, high specificity, high accuracy, high exactness and the like, for fast detecting recessive malachite green residual in the aquatic products.

Description

technical field [0001] The invention belongs to the field of ELISA and veterinary drug residue detection in the field of food safety. Specifically, the present invention relates to an ELISA kit suitable for detecting recessive malachite green residues in aquatic products. technical background [0002] Malachite green (Malachite green) molecular formula is: C 23 h 25 ClN 2 , is a triphenylmethane triphenylmehtane dye for industrial use, because it has the effect of antibacterial and anti-Gram-positive bacteria, and the price is cheap, so it is widely used in fish farming to prevent saprolegniasis, gill mold, and small melon bug disease, etc., and in order to prolong the life of fish with damaged scales, malachite green is often used during transportation and in storage ponds. When the aquaculture water contains malachite green, fish, shrimp, etc. will quickly absorb malachite green and then metabolize into recessive malachite green (Leμcomalachite green, LMG), that is, N,...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
Inventor 杨晓慧张猛陈实平张波石立杰黄启明王晓航熊勇华
Owner WUXI ZODOLABS BIOTECH
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