Enrofloxacin monoclonal antibody and application

A monoclonal antibody, enrofloxacin technology, applied in the field of immunochemistry, can solve the problems of low specificity and high cross-reaction rate, and achieve the effect of rapid and sensitive detection

Inactive Publication Date: 2009-10-28
泰州市蛋白质工程研究院
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The monoclonal antibody has high selectivity to fluoroquinolones, and the cross-reaction rates with ciprofloxacin, marbofloxacin, sarafloxacin, and dafloxacin were 110.84%, 27.40%, 71.05%, and 37.41%, respectively. %, the cross-reaction rate is relatively high, and the specificity is not strong

Method used

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  • Enrofloxacin monoclonal antibody and application
  • Enrofloxacin monoclonal antibody and application
  • Enrofloxacin monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Reagent and material preparation

[0031] Standard enrofloxacin (China Veterinary Drug Administration), bovine serum albumin (BSA) (Amresco, 0332), human serum albumin (HSA) (Sigma, A9511), ovalbumin (OVA) (Sigma, A5503 ), carbodiimide hydrochloride (EDC) (Sigma, E6383), N-hydroxysuccinimide (NHS) (Sigma, 130672), N, N-dimethylformamide (DMF) (Sigma, D4551 ), Freund's complete adjuvant (Sigma, F5881), Freund's incomplete adjuvant (Sigma, F5506), tetramethylbenzidine (TMB) (Amresco, 0759), HAT (Sigma, H0262) and HT (Sigma, H0137), sodium chloride (Amresco, 0241), potassium chloride (Amresco, 0395), potassium dihydrogen phosphate (Sigma, P9791), disodium hydrogen phosphate (Sigma, 71639), goat anti-mouse IgG-HRP (Jackson, 115-035-044), dimethyl sulfoxide (DMSO) (Applichem, 0231), polyethylene glycol 4000 (PEG4000) (Sigma, P7306), DMEM high glucose medium (Gibco, 11995), fetal bovine serum ( Gibco, C2027050).

[0032] Experimental animals and cells: Balb / c mice (6-8 wee...

Embodiment 2

[0122] drug cross reaction test

[0123] The monoclonal antibody of enrofloxacin and enrofloxacin hapten structural analogues such as ciprofloxacin hydrochloride, ofloxacin, and mesylate Flufloxacin, pefloxacin mesylate, difloxacin hydrochloride, norfloxacin, lomefloxacin for competition experiments, dilute these standards into different concentrations for indirect competition ELISA, draw inhibition curves, and calculate competitors IC 50 value and cross-reactivity rate, the highest cross-reaction rate was 1.464%. The monoclonal antibody of enrofloxacin reported in the literature has a cross-reaction rate of 110.874 with ciprofloxacin, a cross-reaction rate with sarafloxacin of 71.05%, and a cross-reaction rate with other quinolones of 20%. Above, and the anti-enrofloxacin monoclonal antibody prepared by the present invention only reacts with enrofloxacin, and the cross-over rate of reacting with other quinolones is below 1.5%, therefore, the anti-enrofloxacin prepared in th...

Embodiment 3

[0127] This example is an example of the application of monoclonal antibody 8E6' in the establishment of an ELISA method for the detection of enrofloxacin residues in the present invention, which can be used for the detection of enrofloxacin residues in chicken, aquatic products, milk and honey. Taking chicken (purchased in the market) as an example, the main steps for detecting enrofloxacin residues in chicken are as follows:

[0128] 1. Sample pretreatment: Weigh 15g chicken sample for homogenization, weigh 0.5g homogenate, add 1.5ml 80% methanol solution diluted with sample diluent, shake and mix for 30min, centrifuge at 2000g for 10min, take 100μL Mix the supernatant with 900 μL sample diluent, and take 50 μL for detection.

[0129] 2. The detection principle of the kit in the present invention is the indirect competition ELISA method. The original OVA-ENR coating is coated on a microporous plate, and serially diluted enrofloxacin standards or samples are added, and then a...

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Abstract

The invention relates to an enrofloxacin monoclonal antibody and application, relates to hybridoma strains thereof, and belongs to the technical field of immunochemistry. The enrofloxacin monoclonal antibody is generated by mouse hybridoma strains 6A4 and 8E6. The preparation method comprises the following steps that: enrofloxacin and carrier proteins BSA, HAS and OVA are coupled by a carbodiimide method to synthesize artificial immunogens EnR-BSA, EnR-HSA and coatingen EnR-OVA; a Balb / c mouse is immunized by the synthesized artificial immunogens EnR-BSA and EnR-HSA; a spleen cell of the immunized mouse is extracted to be fused with a SP2 / O myeloma cell and coated by the coatingen EnR-OVA; indirect ELISA method and indirect competition ELISA method are established to screen the hybridoma strains which can stably secrete specific antibody; the obtained cell strain immunized Balb / c mouse is used to prepare ascites; the ascites is purified by a caprylic acid-ammonium method and an ion exchange method; and valences of antibodies of two purified cell strains reach over 1.024*10 and 1.28*10. The monoclonal antibody has strong specificity, can be applied to preparation of enrofloxacin residue inspection kit and aerosol test strip, and can sensibly and quickly inspect the enrofloxacin residue.

Description

technical field [0001] The invention relates to a specific monoclonal antibody of enrofloxacin, its preparation method and application, and also relates to its hybridoma cell line, which belongs to the technical field of immunochemistry. Background technique [0002] Enrofloxacin (enrofloxacin) is the first fluoroquinolone drug specially used for livestock and poultry. It is widely used in veterinary clinical prevention and treatment because of its advantages such as broad antibacterial spectrum, strong antibacterial activity, easy absorption, and wide distribution in the body. With the wide application of this drug in food-borne animals, the toxic and side effects caused by it, such as neurotoxicity, renal function damage, allergic reaction, and arthritis in young animals, are also increasing. The discovery of the problem of drug resistance induced by human pathogenic bacteria due to the residue of the drug in animal food has further deepened people's understanding of the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C12N15/06C12P21/08G01N33/577
Inventor 唐宏王晓艳
Owner 泰州市蛋白质工程研究院
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