Development Of Polyclonal Antibody And Immunoassay For Alternariol

Alternariol(AOH),which is the secondary metabolite of genus Alternaria,are considered as a kind of mycotoxin with genotoxic, estrogenic, and mutagenic properties.Recently, natural occurrence of alternariol has been reported in abundant granis, fruits and vegetables, causing a serious damage to humanity. Therefore, it's essential to raise awareness of the detection for AOH in agricultural products. Nowadays, most analytical approaches applied to the alternariol determination in foodstuff are liquid chromatography tandem mass spectrometry that is accurate and relable with a trouble of expensive maintenance cost, proficient experts, complicated pre-treatment and low throughout. In contrast, immunoassay, as a rapid, sensitive, and high throughout analytical technique,could deal with the on-site screening for a wide range of food commodities coupled with instrument validation techniques. In this study, several haptens were synthesized to the preparation of high-affinity polyclonal antibodies against alternariol, Meantime, two rapid immunoassay analytical methods(ic ELISA and ic CLEIA) were established. The following are the main contents:Based on the acquisition of Alternariol, several haptens attacached to the proteins by means of active ester method, N, N-carbonyldiimidazole catalyst and mannich reaction were designed to obtain four immunogens(AH1-BSA, AH2-BSA, AM-BSA, AC-BSA).After antisera screening, two antiseras which immunized by AH2-BSA and AC-BSA show no specificity against AOH, while others exhibit a high affinity. The titer of AH2, AC antiserum were 1:64000 and 1:32000, and the inhibition rate to 1 ?g / m L of AOH were91% and 37%.A indirect competitive enzyme-linked immunoassay(ic ELSA) for the rapid determination of AOH was developed. The half maximal inhibitory concentration(IC50) of the established method was 2.62 ?g/L. The limit of detection was 0.31 ?g/L with a linear range between 0.46 ?g/L and 15.8 ?g/L, and the cross-reactivity of the seven analogues waslower than 0.1%. The recovery of the spiked samples was between 77.24% and 115.37%after the optimization of the pre-treament and the elimination of the matrix interference. This proposed ic ELISA provided a valid method for a rapid detection for AOH in real samples.A indirect competitive chemiluminescene enzyme immunoassay(ic CLEIA) for the rapid determination of AOH was developed. The half maximal inhibitory concentration(IC50) of the established method was 0.37 ?g/L. The limit of detection was 0.068 ?g/L with a linear range between 0.11 ?g/L and 9.12 ?g/L, and the cross-reactivity of the seven analogues was lower than 0.1%. The recovery of the spiked samples was between 72.67%and 115.80% after the optimization of the pre-treament and the elimination of the matrix interference. This proposed ic CLEIA provided a valid method for an rapid detection for AOH in real samples.