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Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet

A technology of colorless malachite green and enzyme-linked immunosorbent reagents, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex hapten synthesis methods and the inability to detect metabolite metabolites, so as to achieve less health hazards, High sensitivity and good solubility

Inactive Publication Date: 2012-07-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The Chinese patent with application number 200810202733.8 discloses an ELISA reagent and method for detecting malachite green. The invention adopts active ester method to couple malachite green hapten with keyhole limpet hemocyanin and bovine gamma globulin respectively. Immunogen and coating original, the obtained ELISA reagent and method can only detect malachite green, but cannot detect its metabolites and metabolites of crystal violet
Another Chinese patent with the application number 200910058317.X discloses an enzyme-linked immunosorbent assay method for the determination of the total amount of malachite green and colorless malachite green in water samples and aquatic products, which is characterized by the synthesis of amino colorless malachite green As a hapten, it is coupled with bovine serum albumin and ovalbumin to obtain an immunogen and a coating source, and polyclonal antibodies are obtained by immunizing animals. The synthesis method of the hapten used in this invention is complicated and involves relatively dangerous hydrogenation. reaction system

Method used

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  • Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet
  • Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet
  • Monoclonal antibody, ELISA method and kit used for detecting malachite green, leuco malachite green, and leuco crystal violet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of hapten

[0034] 1.1 Synthesis of p-[3-(ethylcarboxy)propoxy]benzaldehyde

[0035] Drying treatment: take a bottle of N,N-dimethylformamide (DMF) 500ml and add anhydrous sodium sulfate 60g, shake it, let it stand overnight, filter under reduced pressure, collect the dried DMF in a dry 1000ml one-mouth bottle, seal it , store in a dry and cool place for later use.

[0036] Drug weighing: Accurately weigh 34g of 4-hydroxy-benzaldehyde, 28g of potassium carbonate, and 68g of ethyl 4-bromobutyrate.

[0037] Hydroxyalkylation reaction: Add the above three medicines weighed together into 200ml of dry DMF, and reflux at 110°C overnight. Let it stand at room temperature, filter, and extract the filtrate with ethyl acetate, and dry it in vacuum. A colorless oily liquid with fruity fragrance is obtained, which is p-[3-(ethylcarboxy)propoxy]benzaldehyde.

[0038] 1.2 Synthesis of p-[3-(ethylcarboxy)propoxy]leucomalachite green

[0039] Weighing: Accura...

Embodiment 2

[0045] Example 2 Preparation of Immunogen and Coating Gen

[0046] 2.1 Activation of drug carboxy terminus

[0047]Drug weighing: Accurately weigh 80 mg of LMG-HEO, 20 mg of 1,3-dicyclohexylcarbodiimide (DCC), and 11.5 mg of N-hydroxysuccinimide (NHS).

[0048] Activation of the carboxyl terminal of the drug: put the three drugs weighed above into a 10mL reaction bottle, add DMF3mL and a magnetic stirrer, stir and react at room temperature in the dark for 16 hours, filter, and take the filtrate (this is liquid A) for later use.

[0049] 2.2 Preparation of carrier protein solution

[0050] Weighing: Accurately weigh (measure) 16mL of 0.1M sodium bicarbonate solution with pH 8.5, 100mg of human serum albumin (HSA) or 100.mg of ovalbumin (OVA).

[0051] Preparation of liquid B: Dissolve 100 mg of HSA into 16 mL of 0.1M sodium bicarbonate solution at pH 8.5, add a magnetic stirrer, and this is liquid B.

[0052] Preparation of liquid C: Dissolve 100.mg of OVA into 16 mL of 0.1M...

Embodiment 3

[0056] Example 3 Preparation of Monoclonal Antibody

[0057] Preparation of hybridoma cells: refer to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press, 2001 Edition: Immunize Balb / C mice with the immunogen LMG-HEO-HSA prepared in Example 2, and the immunization procedure is: basic Immunization After emulsifying the immunogen with an equal volume of Freund's complete adjuvant, inject it subcutaneously at multiple points on the back of the mouse. The interval between the first and second immunizations is 3 weeks, and then boost the immunization every 2 weeks, and emulsify with incomplete adjuvant , and finally intraperitoneally injected three days before the fusion to strengthen the immunization, the amount of antigen was doubled, and no adjuvant was added. At the time of fusion, a Balb / C mouse that had undergone the final booster immunization was taken, sacrificed by bleeding from the eye socket (serum was collected, it was a positive s...

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Abstract

The invention discloses a monoclonal antibody which can be used for identifying malachite green, leuco malachite green, and leuco crystal violet. The invention also discloses an ELISA method and a kit used for detecting malachite green, leuco malachite green, and leuco crystal violet. The monoclonal antibody provided by the invention is secreted by a hybridoma cell 4B10. The hybridoma cell is collected in China Center for Type Culture Collection with a collection number of CCTCC NO: C201143. The ELISA method provided by the invention comprises the steps of immunogen preparation, coating antigen preparation, antibody preparation, sample processing, sample detection, and the like. Compared to prior arts, the monoclonal antibody prepared by the invention can be used for identifying leuco crystal violet, malachite green, and leuco malachite green at a same time. The method and the kit provided by the invention are convenient, fast, sensitive, and accurate.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of recognizing malachite green, colorless malachite green and colorless crystal violet and an ELISA method for detecting malachite green, colorless malachite green and colorless crystal violet with kit. Background technique [0002] Malachite green and crystal violet belong to triphenylmethane dyes, which can be used as bactericides and insect repellents, and are the most effective antibacterial dyes in medicinal dyes. Because malachite green and crystal violet are potentially carcinogenic, they have been banned in many countries, but there are still illegal uses, so it is necessary to strengthen the inspection and monitoring of the drug. [0003] Malachite green and crystal violet can be metabolized into colorless malachite green and colorless crystal violet respectively. At present, the method for detection of malachite green, crystal violet and their metabolite residues is mainly high-performan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543
Inventor 袁宗辉翟长友彭大鹏王玉莲潘源虎黄玲利陈冬梅陶燕飞戴梦红刘振利
Owner HUAZHONG AGRI UNIV
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