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Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof

An aminohydantoin and monoclonal antibody technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the trouble of standardization of detection technology, can not achieve standardized production, can not reach zero Residue detection requirements and other issues have been achieved to achieve the effects of low toxicity, high synthesis efficiency, and simple synthesis process

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xu et al. (2009) did not directly report the IC of the AHD polyclonal antibody used 50 Value, the colloidal gold detection test strip method detects AHD residues in pig urine with a detection limit of 10 μg / L, which is higher than MRPL and cannot meet the requirements of zero residue detection
In addition, although polyclonal antibodies have high sensitivity, they have individual differences and cannot achieve long-term continuous standardized production, which brings troubles to the standardization of detection technology.

Method used

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  • Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof
  • Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof
  • Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Synthesis of Haptens

[0056] Nitrofurantoin residual marker AHD reacted with p-carboxybenzaldehyde (4-CBA) in the medium of triple distilled water and N,N-dimethylformamide. The specific process was as follows: Weigh AHD·HCl 0.60g and 4-CBA 0.30 g, add 6mL triple distilled water and DMF to dissolve respectively, and use Na 2 CO 3 After adjusting the AHD aqueous solution to neutrality, slowly add it to the 4-CBA solution, and react at 60°C to 70°C for 4h. 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 CPAHD.

Embodiment 2

[0057] Example 2: Synthesis of Complete Antigen

[0058] Synthesis of immunogen: CPAHD and bovine serum albumin were coupled to synthesize immunogen by the mixed anhydride method. The specific process was as follows: Weighed 20 mg of CPAHD, dissolved it in 5 mL of N, N-dimethylformamide, stirred and added 25 μL of three With n-butylamine, react at room temperature (20-25°C, the same below) for 10 minutes. The above solution was pre-cooled in an ice bath, and 44 μL of isobutyl chloroformate was added. React at room temperature for 1 to 1.5 hours to obtain an active intermediate product. Weigh 132mg of bovine serum albumin (BSA) and dissolve it in 10mL of phosphate buffered saline (pH 7.4). Add the active intermediate product dropwise to the bovine serum albumin solution, and react at room temperature for 4-5 hours. Dialyze against phosphate buffer (pH 7.4) at 4°C for 3 days. Aliquot and freeze-dry to obtain the immunogen, named CPAHD-BSA, and store at -20°C.

[0059] Coati...

Embodiment 3

[0060] Embodiment 3: Preparation of monoclonal antibody

[0061] 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 CPAHD-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, take a Balb / C mouse that has undergone the final booster immunization, sacrifice it by bleeding from the eye socket (collect the serum, it is positive serum), and immerse it in 75% alcohol for 5 minutes for disinfection.

[0062] ...

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Abstract

The invention discloses a monoclonal antibody of furantoin residue marker aminohydantoin, and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell AHD / 3D4 of which the collection number is CCTCC NO:C201151. The preparation method comprises the following steps: A, coupling hapten CPAHD and bovine serum albumin to obtain immunogen; B, coupling hapten CPAHD 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 AHD / 3D4 of which the collection number is CCTCC NO:C201151; 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 furantoin 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 1-aminohydantoin residue in animal edible tissues.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of detecting nitrofurantoin residue marker 1-aminohydantoin (AHD), and also relates to a method capable of detecting nitrofurantoin residue marker 1- The preparation method of the monoclonal antibody of aminohydantoin (AHD) also relates to the use of a monoclonal antibody capable of detecting nitrofurantoin residue marker 1-aminohydantoin (AHD). Background technique [0002] Nitrofurantoin belongs to nitrofuran drugs and has a basic structure of 5-nitrofuran ring. It is a broad-spectrum antibacterial drug and is effective against Gram-positive bacteria, Gram-negative bacteria, fungi, and protozoa. widely used in industry. Toxicological tests have proved that it has serious toxic effects such as carcinogenicity and mutagenesis, and its toxicity in nitrofuran drugs is second only to nitrofurazone. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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 HUAZHONG AGRI UNIV
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