Establishment Of Immunoassay For Bisphenol A Diglycidyl Ether And Its Derivatives In Canned Food

Bisphenol A diglycidyl ether（BADGE）is a light yellow liquid epoxy compound,which was synthesized by the condensation of epichlorohydrin（ECH）and bisphenol A（BPA）.It is widely used as the basic material for the polymerization of epoxy resins and mainly used as an interior coating for metal canned foods,the purpose of which is to prevent direct contact between the food contents and the metal material.During the processing and storage of canned foods,the monomeric compound BADGE contained in the interior coating will gradually migrate into the food,and will undergo hydrolysis or chlorination upon contact with the food to produce derivatives such as BADGE·H₂O,BADGE·HCl and BADGE·HCl·H₂O.These substances,when ingested by the body through the food chain,can cause abnormalities in the endocrine and nervous systems of the body,resulting in adverse health effects.In this study,an indirect competitive enzyme immunoassay method（ic-ELISA）and Au NPs immunochromatographic method were developed for the detection of residues of BADGE,BADGE·HCl,BADGE·H₂O and BADGE·HCl·H₂O in canned foods based on broad-spectrum polyclonal antibodies.The main studies and results are as follows:（1）The hapten-C and hapten-F were designed and synthesized based on the characteristic structures of BADGE,BADGE·HCl,BADGE·H₂O and BADGE·HCl·H₂O.Immunogens and coating antigens were prepared by coupling haptens to carrier proteins using the EDC and DCC methods,and eight antisera were obtained by immunizing New Zealand white rabbits.The sensitivity of eight antisera against BADGE and its derivatives were analyzed under homologous and heterologous coating.Two kinds of antisera were selected and purified by affinity chromatography to obtain PAb-1 and PAb-2 antibodies at the concentrations of 2.17and 1.94 mg/m L,respectively,and an indirect competitive ELISA method was established to detect BADGE and its derivatives based on these two antibodies.（2）An indirect competitive ELISA method was established for detection of BADGE and its derivatives in canned lunch meat.The effects of homologous coating and heterologous coating on the sensitivity of the method were analyzed,and the experimental parameters were optimized.The optimum experimental conditions were determined as follows:when antibody was PAb-1 at the dilution of 400-fold,the coating antigen was DCC-F-OVA at the concentration of 0.375μg/m L.When antibody was PAb-2 at the dilution of 200-fold,the coating antigen was EDC-C-OVA at the concentration of 1.5μg/m L.The dilution factor of the HRP-labeled secondary antibody was 20000-fold for the detection of BADGE and BADGE·HCl·H₂O,and the dilution factor of the secondary antibody was 40000-fold for the detection of BADGE·H₂O and BADGE·HCl.The blocking solution was 3%skimmed milk powder dissolved in PBS.Under the optimal experimental conditions,the sensitivities(IC₅₀)of BADGE,BADGE·HCl,BADGE·H₂O and BADGE·HCl·H₂O were 16.48,20.21,14.46 and 33.76 ng/m L respectively,and the limit of detection(IC₁₅)were 0.73,0.78,0.39 and 1.45 ng/m L respectively.The established ic-ELISA also applied for sample analysis,and the recovery of BADGE and its derivatives at various spiking levels in canned luncheon meat samples ranged from 76.70 to 102.85%.The results detected by the proposed ic-ELISAs were also validated by HPLC analysis,which showed a good agreement.（R²=0.9839）.Canned luncheon meat,canned yellow peaches and red bull beverage were selected to represent three different types of canned foods of animal,plant and beverage origin,respectively.To investigate the effects of storage time and storage temperature on the migration of BADGE and its derivatives in these three types of canned foods,the total migration of BADGE and its derivatives in canned foods were detected by the established ELISA method.The results showed that the total amounts of BADGE and BADGE·HCl,BADGE·H₂O and BADGE·HCl·H₂O in this three canned foods presented an increasing trend with the increase of time and temperature.The results of ic-ELISA were also in good correlation with the results validated by HPLC.（3）Gold nanoparticles（Au NPs）were synthesized by trisodium citrate reduction,and Au NPs were conjugated with PAb-2,which were used as the immunodetection probe for Au NPs immunochromatography.The p H value,the amounts of antibody for conjugation,concentration of coating antigen in T-zone and amount of Au NPs-Abs were optimized.Under the optimal conditions,Au NPs-PAb immunochromatography was established to detect the total amount of BADGE,BADGE·HCl,BADGE·H₂O and BADGE·HCl·H₂O in canned food.The quantitative analysis could be achieved by software analysis with the detection limit of 1 ng/m L.The Au NPs-PAb immunochromatography was also used for the spiked recovery analysis of three canned foods.The obtained recoveries ranged from 87.35 to 100.43%,which was well correlated with the results by HPLC（R²=0.9792）.