Ultrasensitive Immunoassay Of Ochratoxin A And Aflatoxin B₁

PurposeThe aim of this work is to provide a highly sensitive testing method for the determination of ochratoxin A (OTA) and aflatoxin B₁(AFB₁)by adopting the technology of time-resolved fluoroimmunoassay (TRFIA). This work develop rapid and sensitive OTA-TRFIA and AFB₁-TRFIA immunoassay kits that have our own independent intellectual property rights, so as to enhance the level of toxin's immunoassay of our country, meet the urgent needs regarding inspection of agricultural commodities and challenge the international technical barriers.Method1. The immunogen– conjugation of OTA with KLH were prepared. Rabbits were immunized using OTA– KLH to produce polyclonal antibodies. Mice were immunized using OTA– BSA to produce monoclonal antibodies. Optimum OTA-BSA concentration for coating microplate, labelling condition, assay procedures were select, The enhancement solution and washing solution were prepared; The fully automatic manipulation software were compiled; In indirect OTA- TRFIA format by polyclonal antibodies, goat anti rabbit IgG- Eu³⁺ conjugate was used to enable detection. In indirect OTA- TRFIA format by monoclonal antibodies, goat anti mice IgG- Eu³⁺ conjugate was used to enable detection. The OTA-TRFIA kits were developed. The sensitivity, precision, accuracy, recovery rate, specificity and stability of the OTA-TRFIA kits were examined in accordance with the requirements of labelling immunoassay reagent. The OTA-TRFIA kits were compared with the imported ELISA kit. The kits were used to verify the validity of internal quality control samples.2. Rabbits were immunized using AFB₁-BSA to produce polyclonal antibodies. In indirect AFB₁-TRFIA format, aflatoxins B₁-horseradish peroxidase(AFB₁-HRP) coating on wells , a goat anti-rabbit IgG Eu³⁺ conjugate was used to enable detection . The AFB₁-TRFIA kit was developed;The sensitivity, precision, accuracy, recovery rate, specificity and stability of the AFB₁-TRFIA kit were examined. The AFB₁-TRFIA kit was compared with the ELISA kit.Results1. The immunogen --OTA-KLH was prepared. The polyclonal antibodies and monoclonal antibodies were got. A goat anti mice IgG- Eu³⁺ conjugate and goat anti rabbit IgG- Eu³⁺ were prepared by DTTA method .A fully automatic analysis software was compiled; OTA-TRFIA and AFB₁-TRFIA kits were developed and examined respectively.2. The results show that in the OTA-TRFIA by polyclonal antibodies, the OTA detection limit was 0.02μg/L for indirect competitive TRFIA formats. The assay ranges from 0.02μg/L to 400μg/L. The within-run and between-run CVs of the OA-TRFIA were 2.6% and 5.2%...