Development Of Monoclonal Antibody Against The Metabolite AHD Of Nitrofurantoin And Study On Its Rapid Detection Technology

Nitrofurantoin, belonging to nitrofuran drugs, is a kind of broad spectrum antibiotics.Because of its high efficiency and low price, it was widely used in medicine, animal husbandryand aquaculture. But the research showed that the drug has adverse effects to animals andpotentially car-cinogenic, teratogenic and mutagenic toxicity.The drug was banned to use inanimal breeding process in European Union since1995, and our country also had prohibited theuse of the drug since2002. Although our country increased monitoring strength on detectingnitrofurantoin residues in animal products, the phenomenon of nitrofurantoin residue was stillserious. At present the immunological method is the main one for screeing veterinary drug residue,one of the most common is enzyme league immunoassays. However,few papers about ELISAmethod had been reported for detecting nitrofurantoin residues. Chemical luminescence immuneanalysis （CLIA） is an enzyme league immuno-assays combined with chemiluminescence analysis.Because of higher sensitivity and wider linear range, it will be a new trend of immunoassaysdevelopment. Now there is no report about the CLIA in detecting nitrofurantoin residue in China.This experiment would prepare monoclonal antibodies of anti-nitrofurantoin, then the enzymeleague immune test method and chemical luminescence immune detection method would beestablished on the basis of the antibodies.It will take a basis for further development ofnitrofurantoin residue fast detection kit. Specific experiment content as follows:Nitrofurantoin metabolites1-Aminohydantoin hydrochloride （AHD）, derived with4-Formylbenzoicacid, was coupled with bovine serum albumin （BSA） by Mixture anhydrides meth-od.The artificial immune antigen（CPAHD-BSA） was confirmed to be synthetized successfully byultraviolet （UV） and degenerative gel electrophoresis （SDS-PAGE）. The poly-clonal antibodywas produced by the immunization of BALB/c mice with CPAHD-BSA. The titer of mice serumwas1:32000by indirect enzyme linked immunosorbent assay （ELISA） and the specialty wasidentified by indirect competitive ELISA.The antibody was highly specific to AHDderivatives（NPAHD）, the LC₅₀of NPAHD is30.63μg/L and had no crossing reaction with othernitrofuran drugs and their metabolites. The monoclonal antibodies of antinitrofurantoin wasproduced by BALB/c mice immuned with CPAHD-BSA. Two cell line strains of anti-4-CPAHDantibody were obtained and named1E5,2A2, respectively.1E5cell line was better by comparingthe two antibodies specificity.With synthetic AHD-OVA as coating antigen and nitrofurantoin metabolites monoclonalantibody （1E5cell lines）, ELISA test technology was developed, and the results showed that thebest working concentration of coating antigen and antibody were0.5μg/mL,1:5x10⁵, respective-ly. The range of linearity was from0.78to50μg/L, and the LC₅₀was2.74μg/L, the lowest detection limit was0.11μg/L, the regression equation is y=-0.3699x+0.6619,R²=0.9905, the introand inter variation coefficient of ELISA standard curve were2.82%and3.44%respectively, andrecovery results of fish tissue spiked with AHD was between60%⁹0%.Based on the existing enzyme chemiluminescence detection platform developed in ourlaboratory, CLIA methods for detecting nitrofurantoin was established.From0.2to12.5μg/L range, the curve is in a linear relationship, regression equation is y=0.2765x+0.5131, R²=0.9912, the LC₅₀was1.11μg/mL. The intro and inter variation coefficient of CLIA standard curvewere2.82%,3.44%, and the lowest detection limit was0.03μg/L.Recovery results of fish tissuespiked with AHD was between60%⁹0%.