Malachite green vestigial ELISA detection kit and usage method thereof

An enzyme-linked immunoassay and enzyme-linked immunoassay reagent technology, which is applied in the field of malachite green residual enzyme-linked immunoassay kits, can solve the problems of tediousness, difficulty in meeting the needs of practical applications, and many steps

Active Publication Date: 2008-07-23
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Retrieval of relevant literature and patents found that there are few methods and kits for malachite green immunoassay in China, and a Chinese patent "Malachite Green Indirect Competition ELISA Detection Kit in Aquatic Products" (application number 200510060995.1) was retrieved. A malachite green enzyme-linked immunosorbent assay kit, the detection principle adopted by this kit is that the enzyme plate is coated with malachite green antigen, the sample and the coated antigen compete to bind to the malachite green antibody, and the unreacted reagent is washed to remove, Then add enzyme-labeled anti-antibody reaction, wash again, add substrate for color development, and read after termination; due to the use of indirect competition ELISA detection mode, the kit has complicated operation, many steps, long detection time, high production and storage costs, Difficult to meet the needs of practical applications
[0007] In short, the detection methods used in the existing enzyme-linked immunosorbent assay kits are complex and cumbersome, and it is difficult to apply them in practice, and the existing technologies are generally poor in stability, complicated in sample pretreatment and detection steps, high in equipment requirements, and expensive. Insufficient, seriously affected the malachite green residual detection and monitoring, so the development of high stability, simple operation, low equipment requirements, cheap malachite green ELISA kit has very important economic and social significance

Method used

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  • Malachite green vestigial ELISA detection kit and usage method thereof

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Experimental program
Comparison scheme
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Embodiment 1

[0079] The preparation of embodiment 1 antigen

[0080] Preparation of malachite green antigen:

[0081] a. 200mg of purified amino-colorless malachite green plus 0.3mol / L HCL 12mL, stir and dissolve the purified amino-colorless malachite green;

[0082] b. Slowly add 10g / L NaNO under the condition of stirring in an ice bath 2 , to diazotize the amino colorless malachite green while monitoring NaNO 2 Whether it is excessive, the detection method: take the starch / potassium iodide solution and drop it on a white dry glass sheet, add 1 drop of the above diazotization solution, and the diazotization of amino colorless malachite green will be completed within 30 seconds after mixing;

[0083] c. Continue to react the above-mentioned diazotized amino colorless malachite green for 20 minutes, weigh 800 mg of BSA, dissolve it in carbonate buffer to make a 20 g / L protein solution, and slowly add diazotization under the condition of stirring in an ice bath Amino colorless malachite g...

Embodiment 2

[0085] The preparation of embodiment 2 antibody

[0086] Preparation of malachite green mouse monoclonal antibody:

[0087] Animal immunization procedure: Balb / c mice were used as immunized animals, and the conjugated malachite green hapten and bovine serum albumin was used as the immunogen, and the immunization dose was 60 μg / mouse. The emulsifier was mixed with complete Freund's adjuvant and injected intraperitoneally. The same dose of immunogen and the same amount of Freund's incomplete adjuvant were mixed and emulsified at intervals of 3 weeks for a booster immunization. After the fourth immunization, the abdominal cavity was boosted once. Splenocytes were collected 3 days later .

[0088] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 4:1. Cell supernatants were measured by indirect competitive enzyme-linked immunosorbent assay, and positive wells were screened. The positive wells were cloned by microclo...

Embodiment 3

[0091] The extraction of embodiment 3 horseradish peroxidase or alkaline phosphatase

[0092] 1. Extraction of horseradish peroxidase

[0093] a. Water extraction: take by weighing 20 kilograms of washed fresh horseradish or horseradish skin, cut into small pieces, and mince in a pulverizer. Add 10 kg of water to the crushed slag slurry, stir and extract at low temperature for 8 hours, centrifuge at 3000 rpm for 10 minutes, and collect the supernatant.

[0094]b. Fractional separation of ammonium sulfate: add 226 grams of ammonium sulfate powder per liter of filtrate, stir while adding, put overnight at room temperature. The next day, draw the supernatant, and then add 258 grams of ammonium sulfate powder per liter of supernatant, and stir as you add it. After the ammonium sulfate is completely dissolved, put it in a cold room overnight. The next day, the supernatant was sucked off, and the precipitated part was centrifuged at 13,000 rpm for 20 minutes in a refrigerated cent...

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Abstract

The invention discloses an enzyme immunoassay of testing the bice green residues in animal derived food, which comprises an enzyme label plate covering bice green antigen, enzyme label bice green antibody working solution, bice green standard solution, substrate solution, substrate buffer solution, reaction termination solution, concentration washing liquid and sample dilute solution. The invention further discloses a method for using the immunoassay to test bice green residues, which comprises sample pretreatment, testing via the immunoassay, processing and analyzing result. The inventive immunoassay of bice green test uses direct competition enzyme-linked immunoassay adsorption analysis technique, with high sensitivity, high stability, simplified operation, reduced reaction time, reduced error caused by complex operation, reduced cost, wide application for testing samples and high practicality.

Description

technical field [0001] The invention relates to the technical field of enzyme-linked immunoassay detection, in particular to an enzyme-linked immunoassay kit for detecting malachite green residues in animal-derived foods and a method for using the same. Background technique [0002] Malachite green has potential carcinogenic, teratogenic and mutagenic effects, and its use in the aquaculture industry has not been approved by the US Food and Drug Administration (FDA); according to the European Union Act 2002 / 675 / EC, animal-derived The total amount of malachite green and colorless malachite green residues in food is limited to 2 μg / kg; Japan’s positive list also clearly stipulates that malachite green residues shall not be detected in imported aquatic products; my country’s agricultural industry standard "NY5071-2002 No Malachite green is also listed as a prohibited drug in the Guidelines for the Use of Pollution Food and Fish Medicine. [0003] In 2005, malachite green residues...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/535G01N33/577
Inventor 孙远明杨金易沈玉栋王宇雷红涛王弘肖治理
Owner SOUTH CHINA AGRI UNIV
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