Preparation And Application Of Monoclonal Antibodies To Detect The Residues Of Synthetic Stilbenes And Fluoroquinolones In Foods

The use of veterinary drugs in food producing animals for treating or preventing infections has become a major problem to public health.The misuse of non-steroidal stilbenes as growth promoters together with fluoroquinolone as antibiotic in animal livestock has led to a rise in antibiotic and hormonal residues in edible animal food products.In order to protect the consumers,reliable screening methods for rapid,selective and sensitive detection of veterinary chemical residues are necessary to ensure food safety.This essay described the methods for preparation and application of monoclonal antibodies against synthetic stilbenes and fluoroquinolones.Hexestrol?a synthetic hormone derivative of stilbenes?and two fluoroquinolones?lomefloxacin and pefloxacin?were selected as parent haptens.These hapten were covalently coupled either to Keyhole Limpet Hemocyanin?KLH?,bovine serum albumin?BSA?or chicken ovalbumin?OVA?for immunogen synthesis,successful conjugates were confirmed using UV-Vis spectrophotometer and used to immunize the mice for monoclonal antibody?mAb?production.To produce anti-hexestrol monoclonal antibody,the hapten HES-4C was synthesized and covalently coupled to KLH,the obtained immunogen was used to immunize mice.Based on the checkerboard titration,an indirect competitive enzyme-linked immunosorbent assay?ic-ELISA?was developed to detect HES and DES.The half-inhibition concentration(IC₅₀)values of ic-ELISA were 0.15 ng/mL and 0.23 ng/mL for HES and DES,respectively.Milk samples were spiked and analyzed.The recoveries of both synthetic hormones in milk samples were 60.48–102.19%?HES?and 89.34–100.16%?DES?.The immunochromatographic assay,in 0.01 M PBS,showed the visual cutoff values at 0.5 ng/mL for HES and 1.0 ng/mL for DES,which allowed us to detect milk samples of a concentration low to 10 ng/mL for HES and 15 ng/mL for DES.A novel immunogen of lomefloxacin?LFLX-EDEA-BSA?was designed and synthesized using cationized BSA in order to prepare anti-lomefloxacin monoclonal antibody.The Ic-ELISA had an IC₅₀ value of 0.19 ng/mL with a limit of detection of 0.04 ng/mL in 0.01 M phosphate-buffered saline?PBS?.The cross-reactivity of anti-LFLX mAb exhibited high sensitivity with only two FQs with a percentage CR of 111.8%for norfloxacin and 82.6%for enoxacin.The intra-and inter-assay recovery rates of LFLX in bovine milk samples were 98.02–107.40%and 100.65–107.82%.The immunochromatographic assay of LFLX in PBS and spiked bovine milk samples had visual cutoff values at 1 and 5.0 ng/mL,respectively.Lastly,a broad sensitive anti pefloxacin?PEF?monoclonal antibody?mAb?,indirect competitive enzyme-linked immunosorbent assay?ic-ELISA?and lateral flow test strip methods were developed to detect nine fluoroquinolone?FQ?residues in chicken muscle samples.Under optimized conditions of ic-ELISA,the anti PEF mAb showed reasonable cross-reactivity with nine FQs with a limit of detection of 0.082 ng/mL.The recoveries from spiked samples were within the range of 62.42 to 111.47%and 63.5 to 113.79%respectively for intra and inter-assays.The visual cut off values of the lateral flow test strip in 0.01 M PBS and food matrices were within the range of 2.5 to 50 ng/mL and 5 to 100 ng/mL respectively.In summary,three monoclonal antibodies were successfully produced and were exploited to develop ic-ELISA and immunochromatographic strip devices.The sensitivity and limit of detection of developed immunoassays were suitable for routine monitoring and screening important residues of stilbenes and fluoroquinolones.