The Study On Chemiluminescent Enzyme-linked Immunosorbent Assay For The Determination Of Sulfonamides Residues

An enzyme-linked immunosorbent assay(ELISA) based on polyclonal antibody with enhanced chemiluminescent(ECL) detection of sulfonamides in food samples had been developed.The anti-sulfonamides polyclone antibodies were produced in rabbits.A series of parameters of this method were optimized in this study,including the concentration of coated antibody,concentration of enzyme conjugate and diluent of enzyme conjugate.And the performance of the developed method had also been evaluated systemicallyThe specificity of the ECL-ELISA was evaluated by determining the cross-reactivity with sixteen sulfonamides.ECL-ELISA was about 2～3 times more sensitive compared to colorimetric ELISA.The limits of detections of these sixteen sulfonamides were 0.1～31.0μg/L.The buffer of phosphate buffered saline(PBS) was used to dilute the extract to removal the matrix effects.This ECL-ELISA presented here successfully determined five kinds of sulfonamides(sulfisozole,sulfathiazole,sulfameter,sulfamethoxypyridazine and sulfapyridine) in chicken muscle and pig muscle samples.The recoveries were 62.1～110.3%and most of the coefficients of variation were under 15%.The accuracy and precision of ECL-ELISA was validated by the instrumental method of HPLC.The correlation coefficients(R~2) of the ECL-ELISA and HPLC data were 0.9546 in chicken muscle and 0.9379 in pig muscle.The consistency of ECL-ELISA and colorimetric ELISA was acceptable,and the R~2 was 0.9481.Furthermore,the substrate of chemiluminescent was groped to improve the stability of the experiment.Though the stability of new substrate could sustain about seven days,the result could supply references to the future study.The developed ECL-ELISA could satisfy the maximum residue limit(100μg/L) of the USA and European Union,which provide a base for study the determination methods of other pesticides and animal medicine.