Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof

A technology of monoclonal antibody and nitrofurazone, which is applied in the field of veterinary drug residue analysis and immunology, can solve problems such as the inability to realize standardized production and the difficulty in establishing standardization of detection technology, and achieve the effects of low toxicity, high synthesis efficiency, and simple synthesis process

Active Publication Date: 2013-09-11
WUHAN SHANGCHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the polyclonal antibody has high sensitivity, the individual differences in rabbits lead to defects in the standardized production process, a

Method used

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  • Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof
  • Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof
  • Monoclonal antibody of furacilin residue marker semicarbazide, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Synthesis of Hapten SEM Derivatives (CPSEM)

[0055] React the nitrofurazone residue marker SEM with p-carboxybenzaldehyde (4-CBA) in the medium of triple distilled water and N,N-dimethylformamide, the specific process is as follows: Weigh 0.15g of SEM·HCl and 4- CBA 0.23g, add 4mL triple distilled water and DMF to dissolve respectively, use Na 2 CO 3 After adjusting the SEM aqueous solution to neutrality, slowly add it to the 4-CBA solution, and react at room temperature (20-25°C, the same below) for 3h. After the reaction was terminated, it was filtered with suction and washed three times with triple distilled water and absolute ethanol to obtain a light yellow solid, which was dried and stored in cold storage. This was the hapten CPSEM.

Embodiment 2

[0056] Example 2: Synthesis of Complete Antigen

[0057] Synthesis of the immunogen The immunogen was synthesized by coupling CPSEM and bovine serum albumin by the active ester method, and the specific process was as follows: Weigh 20.7 mg of CPSEM and dissolve it in 2 mL of N,N-dimethylformamide, stir and add N-hydroxyl Succinimide (NHS) 14.4 mg and N, N-dicyclohexylcarbodiimide (DCC) 27.5 mg were stirred and reacted overnight at room temperature (20-25°C, the same below). Centrifuge at 10000r / min for 10min, take the supernatant for later use, this is liquid A. Weigh 167 mg of bovine serum albumin (BSA) and dissolve it in 10 mL of phosphate buffered saline (pH 7.4) for later use. This is liquid B. Add liquid A to liquid B dropwise under magnetic stirring, react in ice bath for 10 h, centrifuge at 10,000 r / min for 15 min, and collect the supernatant. The supernatant was dialyzed against phosphate buffer (pH 7.4) at 4°C for 3 days. Aliquot and freeze-dry to obtain the immuno...

Embodiment 3

[0059] Embodiment 3: Preparation of monoclonal antibody

[0060] Preparation of hybridoma cell lines: 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 CPSEM-BSA conjugate prepared in Example 2, and the immunization procedure is: basic Immunization After the immunogen was emulsified with an equal volume of complete Freund's adjuvant, it was injected subcutaneously at multiple points on the back of the mouse, and the immunization was boosted once every 2 weeks, and emulsified with incomplete adjuvant, and finally injected intraperitoneally three days before the fusion , Enhanced immunization, double the amount of antigen, no adjuvant. At the time of fusion, one Balb / C mouse that had undergone the final booster immunization was taken, sacrificed by bloodletting from the orbit (serum was collected, it was positive serum), and was sterilized by immersing in 75% alcohol by volume for 5 mi...

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Abstract

The invention discloses a monoclonal antibody of furacilin residue marker semicarbazide, and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell SEM/4G6 of which the collection number is CCTCC NO:C201150. The preparation method comprises the following steps: A, coupling hapten CPSEM and bovine serum albumin to obtain immunogen; B, coupling hapten CPSEM and ovalbumin to obtain coating antigen; C, preparing the monoclonal antibody from the immunogen in the step A, wherein the monoclonal antibody is secreted by a hybridoma cell strain SEM/4G6 of which the collection number is CCTCC NO:C201150; D, coating a solid-phase carrier with the coating antigen in the step B; E, treating a sample to be detected with acid, adding benzaldehyde, performing ultrasonic derivation, extracting with ethyl acetate, taking nitrogen gas at the ethyl acetate layer, performing blow-drying, purify n-hexane, and redissolving a sample diluent to obtain a substance to be detected; and F, performing ELISA (enzyme-linked immunosorbent assay) detection on the substance to be detected. The invention also discloses application of a kit in furacilin residue detection of animal edible tissues. The method is convenient, quick, sensitive and accurate, and can be used for developing an ELISA kit capable of detecting semicarbazide residue in animal edible tissues.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and in particular relates to a monoclonal antibody capable of detecting the semicarbazide (SEM), a marker of nitrofurazone residue, and also relates to a monoclonal antibody capable of detecting the semicarbazide (SEM), a marker of nitrofurazone residue. The preparation method of the cloned antibody also relates to the use of a monoclonal antibody capable of detecting the residual marker semicarbazide (SEM) of nitrofurazone. Background technique [0002] Nitrofuran belongs to nitrofuran drugs and has the basic structure of 5-nitrofuran ring. It is a broad-spectrum antibacterial drug and is effective against Gram-positive bacteria, Gram-negative bacteria, fungi, protozoa, etc. It has been used in breeding widely used in the industry. Toxicology tests have proved that nitrofurazone and its metabolites have significant carcinogenic, mutagenic and other seriou...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543C12R1/91
Inventor 袁宗辉柳璇彭大鹏王玉莲陈冬梅陶燕飞黄玲利戴梦红刘振利
Owner WUHAN SHANGCHENG BIOTECH
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