High Sensitive Immunological Detection Technology Of Time-resolved Fluoroimmunoassay To Aflatoxin

Aflatoxin B₁（AFB₁） is produced by partial enterotoxigenic strains of fungisecondary metabolites, which widely exists in all kinds of food and nature, with thestrongest toxicity. Aflatoxin B₁can produce the strong poison and strong carcinogenicon human and animal’s liver and kidney.Corn,feed and soybean meal mainly pollutedby AFB₁,which can cause many pathological changes of people and animals, and evendeath. The International Agency for Research on Cancer（IARC） designated AFB₁asclass I carcinogen. To control AFB₁producing pollution to the human’s food andanimal’s feed from various aspects strict, minimize the dangers of AFB₁, developmentof new high sensitive method of AFB₁detection is particularly important. With thecontinuous development of technology, the time-resolved fluorescence immunoassaymethod （TRFIA） obtained fast development. It has no radioactive contamination, highdetection sensitivity, simple operation, good repeatability, and other advantages, inaddition, TRFIA can meet a lot of the testing of the sample. In this study, the structurecharacteristics of AFB₁was analysed, artificial antigen was prepared first, and then themonoclonal antibody obtained by purifing the mice ascites. The indirect competitiveTRFIA AFB₁kit was prepared successfully and its performance was evaluated. A fasthandling of samples with common method was established, and lay the foundation forthe application and promotion of AFB₁rapid detection kit.Using NHS and EDC to couple AFB₁with the carrier BSA protein, and usingultraviolet （UV） scanning method and SDS-PAGE gel electrophoresis and monoclonalantibody to test the artificial antigen. The results showed that the ultraviolet absorptionpeak of artificial antigen contained ultraviolet scanning characteristics of AFB₁andcarrier protein peak, and moved at this base.Its molecular weight is more than carrierprotein, and had good immunogenicity, which fully showed the preparation of AFB₁artificial antigen is successful.Monoclonal antibodies were used to construct an indirect competitive assay for aflatoxin B₁（AFB₁） with time-resolved fluoroimmunoassay（TRFIA）. Anti AFB₁-BSAMonoclonal antibodies were obtained from mices immunized with AFB₁-BSA. AFB₁-BSA was coated onto the microtitre plate and incubated with standard AFB₁or samplesand anti-AFB₁antibody. A goat anti-mice IgG-Eu³⁺conjugate were used. The AFB₁detection limit was0.0094μg/L for competitive TRFIA. The detection range of AFB₁was from0.01to10μg/L. The intra-assay and inter-assay CV were3.05%and4.71%respect-tively. The mean recovery rate was91.75%. The cross reactivity of AFB₁withaflatoxin B₂was5.12%and didn’t react with OTA and BSA. The results showed thatTRFIA was a sensitive method in detecting. The high sensitivity and wide optimalrange of this method show its great application prospect.