Rapid Enrichment Of Trace Aflatoxin B₁by Immunomagnetic Bead And Detection By ELISA

Aflatoxin B1is a type of toxic secondary metabolite, the International Agency for Research on Cancer classified AFB1as a Group I carcinogen based on sufficient eveidence. Soybean sauce, a traditional fermented food in china has shown existence of different levels of aflatoxin B1pollution. Even at very low concentration, the intake of AFB1over a long time may be highly dangerous. Therefore, it is important to establish a rapid method to detect AFB1, especially in the trace AFB1sample.The conditions of coating and pretreatment of aflatoxin B1were developed and determined, A method was established to detect the aflatoxin B1in soybean sauce using the immunomagnetic beads system for pretreatment and ELISA method for quantification. The overall time including IMS and ELISA was2h. The results demonstrated the pH7.40.02M PB buffer solution as the coating conditions was best, the best ratio of antibody to magnetic beads was55μg/mg, and the20%methanol-PBS as the condition of adsorption had very good adsorption effect. The ELISA method showed a good linear relationship in aflatoxin B1concentration of0.05μg/mL～0.3μg/mL (r2=0.9842). The average recoveries across spike levels varied from0.5μg/kg to7μg/kg were83.6%～104%with relative standard deviation between4.2%and11.7%. Two kinds of direct and indirect immunomagnetic bead methods were evaluated for direct pretreatment of aflatoxin B1in soybean sauce; anti-aflatoxin B1monoclonal antibody was coated with magnetic beads and biotinylated antibody was coated with streptavidin magnetic beads, respectively. The results demonstrated the efficiency of antibody coating with blank beads was greater than that of antibody coating with streptavidin beads. However, the adsorption effect was better with streptavidin beads as the less quantity of IMBs were needed especially in the trace AFB1sample. The0.5mg IMBs of direct method and0.3mg IMBs of indirect method were adopted for soybean sauce pretreatment of5ng AFB1.The soybean sauce pretreatment method of immunomagnetic beads system was compared with conventional extraction and immune-affinity column method. The results showed that the method of IMS needed shortest time for pretreatment and had high recovery especially in trace AFB1content. With the advantages of dynamic thermodynamics reactions, the method of IMS has superiority over other pre-concentration methods. Compared with conventional extraction, the IMS method had higher recovery, shortens time, simple procedure, and utilizes less organic solvent Compared with immunoaffinity column, the method of IMS had low cost, shorten time, simple pretreatment and sensitive. The reliability of the method was verified by LC-MS method.With the advantages of rapid detection, easy operation, simple equipment, sensitivity, accuracy, and high recovery; this method could be well applied in the determination of aflatoxin B1in soybean sauce samples, especially the trace AFB1sample.