Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof
A technology of monoclonal antibodies and fluoroquinolones, applied in the field of immunochemistry, can solve the problems of expensive, unreachable, and unsuitable for popularization of instrumental analysis methods, and achieve rapid and sensitive detection results
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Embodiment 1
[0022] Reagent and material preparation
[0023] Norfloxacin (National Institute for the Control of Pharmaceutical and Biological Products, 130450-200705), DMF (sigma, D4551), sodium acetate (sigma, S2889), acetic anhydride (Sinopharm Chemical Reagent Co., Ltd.), bovine serum albumin (BSA) (Jackson, 001-000-173), ovalbumin (OVA) (Sigma, A5503), 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).
[0024] Experimental animals and cells: Balb / c mice (6-8 weeks old, female) were p...
Embodiment 2
[0109] drug cross reaction test
[0110] The monoclonal antibody of norfloxacin and norfloxacin hapten structural analogue such as ciprofloxacin hydrochloride, ofloxacin, mesylate Flufloxacin, pefloxacin mesylate, difloxacin hydrochloride, enrofloxacin, lomefloxacin for competition experiments, dilute these standard products into different concentrations for indirect competition ELISA, draw inhibition curves, and calculate competitors IC 50 The results showed that the antibody had a high cross-reaction rate with the other five fluoroquinolones tested except for the low cross-reaction rate with dafloxacin and lomefloxacin , see Table 4 for the specific results.
[0111] type
[0112] Table 4
Embodiment 3
[0114] This example is an example of the application of the monoclonal antibody 4G11' in the establishment of an ELISA method for the detection of norfloxacin residues in the present invention, which can be used for the detection of fluoroquinolone residues in chicken, aquatic products, milk and honey. Taking chicken (purchased in the market) as an example, the main steps for detecting fluoroquinolone residues in chicken are as follows:
[0115] 1. Sample pretreatment: Weigh 15g chicken sample for homogenization, weigh 0.5g homogenate, add 1.5ml sample extract, shake and mix, centrifuge, take 100μL supernatant and 900μL sample diluent, mix, take 50μL for detection.
[0116] 2. The detection principle of the kit in the present invention is the indirect competition ELISA method. The original NOR-OVA is coated on a microwell plate, and 50 μL of 10, 5, 2.5, 1.25, 0.625 and 0.313 ng / ml Norfloxacin or the sample, then add 50 μL anti-fluoroquinolone monoclonal antibody 4G11', the re...
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