Rapid Immunological Detection Techniques And Methods For Pigments And Estrogenic Effectors In Foods

With the deepening of toxicology studies, the toxic effects of synthetic pigments and estrogeniceffectors have gradually been recognized. However, food safety incidents about the excessive andillegal addition of synthetic pigments and estrogenic effectors in foods, continue to occur, whichhave attracted much attentions from researchers and consumers. At present, there are still lack ofsimple, rapid, sensitive and accurate detection methods in terms of China’s food safety supervisionfor these synthetic pigments and estrogenic effectors. Therefore, in this project, we produced highspecific monoclonal antibodies and established ELISA methods for the detection of six substancesincluding erythrosine, tartrazine, basic orange II,4-aminoazobenzene, octylphenol anddibutyl-o-phthalate, based on the principles of immunology. The main contents are as follows:A new and innovative erythrosine hapten was achieved by removing the sodium fromerythrosine and coupling with6-aminocaproic acid. The hapten was conjugated to carrier proteinsto give the artificial completed antigens of erythrosine. BALB/c mice were immunized by theimmunogen to produce the positive spleen cells which could secrete antibodies against erythrosine.The erythrosine monoclonal cell lines were obtained after cells fusion of spleen cells and hybridomacells.The ascites monoclonal antibody was collected from the mice using in vivo induction method,in order to establish erythrosine ELISA method. The IC50of erythrosine monoclonal antibody was3.02ng/mL, with a limit of detection (LOD, IC10) of0.75ng/mL, and the linear range was ranged1.27ng/mL to8.49ng/mL. The erythrosine ELISA method was successfully applied to thedetection of grape juice drinks sample through recovery test, in which the measured intra-assay andinter total recovery was all in the range of92.06%-106.20%and the overall coefficient of variationwas in the range of4.38%-13.93%.A study was carried out in order to investigate the influence of haptens on the production ofhigh specific and sensitive antibodies against tartrazine. Through the synthesis of haptens and theimmunization and so on, a super sensitive tartrazine monoclonal antibody1F3was finally produced,and its IC50value was as low as0.11ng/mL. The established ELISA method was further applied tothe recovery experiment of tartrazine in orange juice beverages. The intra-assay recovery rangedfrom84.12%to109.70%, and its coefficient of variation ranged from5.95%to11.26%; while theinter-assay recovery was from73.38%to93.43%, with its coefficient of variation from5.90%to12.35%. Futher more, in order to evaluate the accuracy of this ELISA method, this assay wassuccessfully applied to the detection of an unknown real positive carbonated sample, and theconcentration of this carbonated sample was13456.73ng/mL.Through the analysis of the chemical structure of basic orange II, a new analogue of basicorange II with a four carbon-atom carboxylated chain was designed and synthesized. And the problem of the protection of the amino in the basic orange molecule was also successfully sovled.Finally, the basic orange monoclonal antibody5E12with high specificity was successfully prepared.The optimized IC50was2.13ng/mL, with a limit detection (LOD) of0.50ng/mL. The linear rangewas0.81-6.93ng/mL. The established ELISA method was used for the detection of basic orange IIin commercially available small yellow croaker. The recovery test showed that the total recoveryranged75.52%to112.95%with the total coefficient of variation of4.99%-7.96%, which provedthat the method is very sensitive and reliable.A hapten of aniline yellow was prepared through diazotization reaction between aminobenzoicacid and metaphenylene diamine. The hapten was further used for the immunization of mice andcell fusion. A monoclonal antibody1G3of aniline yellow was produced. Its IC50value was as lowas44.11ng/mL and its LOD (IC10) was5.09ng/mL. There was no cross reaction between basicorange II and other pigments. The ELISA method based on the monoclonal antibody1G3wasemployed to analyze beancurd in a fortification experiment. The total recovery ranged from76.17%to88.26%, and the coefficient of variation ranged from3.75%to13.30%. This indicatd that thismethod can be applied to the rapid and sensitive detection of aminoazobenzene which wasforbidden to add to foods.A similar analogue (named NPA) of nonylphenol was synthesized through six-step chemicalreactions from azelaic acid and azelaic acid dimethyl ester, based on the previous studies. Amonoclonal antibody3C7with high specificity to octylphenol was produced. The IC50was75.83ng/mL and the LOD was6.55ng/mL. And this antibody against octylphenol had no crossreactivities to other phenols. A recovery experiment with water samples from Taihu Lake wasperformed with the ELISA method based on the monoclonal antibody.The total recovery was in therange of79.53%-96.47%, and the coefficient of variation was between4.79%-12.31%.In the current immunoassays for the dection of phthalate ester, the sensitivity of DBP antibodywas still not high. Therefore, the hapten DBAP was successfully synthesized, and a monoclonalantibody3B7against DBP was sucessfully prepared. The IC50of the antibody was33.94ng/mL andthe detection limit (LOD) was3.31ng/mL, no cross-reactivity was observed to the rest of otherphthalates. The ELISA method based on DBP monoclonal antibody was established for thefortification experiment in commercially available alcohol samples with the recovery of80.3%-95.1%.