| Mycotoxins,secondary metabolites produced by fungal during growth,widely exist in food and feed and cause serious damage to the health of humans and animals.Therefore,it is important to establish a sensitive and rapid detection method used for monitoring and regulation of mycotoxins.Immunological detection method is based on the specific binding of antigen to antibody,and exhibits high sensitivity and easy operability.This study aims to establish immunological methods which are suitable for rapid detection of mycotoxins.With the advantages of ELISA method such as short detection time,simple sample pretreatment,low cost,high sensitivity and wide application potentials,this research project has developed an ELISA method suitable for sensitive Aflatoxins B1(AFB1)detection.This study also established a surface plasma resonance sensor method for quantitative detection of aflatoxin with its merits of no label,real-time monitoring and high sensitivity.In addition,this study prepared a zearalenone immune affinity column which can be used for an effective sample pretreatment purification method and achieve high sensitivity.1?The AFB1-BSA conjugate was characterized by MALDI-TOF-MS to verify successful preparation.The titer of AFB1 monoclonal antibody was determined as 1:8 000 by indirect ELISA,and IC50 of 0.224 ng/mL was achieved with an indirect competitive enzyme immunoassay.This antibody showed high cross-reactivity with some compounds structurally similar to AFB1,which might be used for the detection of aflatoxins.The spiked peanut samples were analyzed,and the recoveries were in the range of 103.45-132.29%,the CV was in the range of 0.74-9.07%%,showing good agreement with the result of LC-MS/MS analysis.2?To determine the best conditions of surface plasmon resonance sensor for quantitative detection of AFB1,this study had optimized conditions of the selfassembled monolayer formation on the gold surface(bare gold soaking temperature and time),antigen flow rate,coupling buffer,concentration of antigen and antibody,reaction buffer and elution conditions.The optimal conditions were as follows: gold chip soaked in 1 mM of MUA ethanol solution 12 h in 4 ?,sample flow rate 20 ?L/min,coupling buffer 10 mM,pH 4.5 sodium acetate buffer,the concentration of antigen 30 ?g/m L,the fixed time 12.5 min,HBS buffer as the reaction buffer,the concentration of antibody 15 ?g/mL,the reaction time 15 min,the flow rate 20 ?L/min.The LOD for detection of AFB1 was 0.312 ng/m L,and excellent linearity in the range of 0.625-10 ng/mL was achieved.Cross reactivity with AFB2,AFG1,AFG2,AFM1 was 72%,33%,43%,and 48%,respectively.The results show that this method can be used for the determination of AFB1 and total aflatoxins.3 ? The performance of prepared zearalenone immune affinity column was evaluated.The average relative column capacity and column blank were 580 ng/0.3 mL gel and 0,respectively.95 % of spiked ZEN can be eluted with 1 m L of methanol.Column capacity remained 50 % of the original capacity after 5 cycles of use.It has a higher cross reactivity towards zearalenone compared with the other five analogues,and the cross reaction rate of ZAN,?-ZOL,?-ZOL,?-ZAL,?-ZAL was 68.4%,63.6%,113.9%,74.4%,and 90.1%,respectively.These results indicate that the column can be used for the pretreatment of zearalenones and related mycotoxins.When the added concentration of ZEN was 200 ng/g,500 ng/g,and 1000 ng/g in the corn samples,the recovery rate was 90.87-121.65% and the CV was 7.00-14.27%.It turned out that the immunoaffinity column can be used in sample purification. |
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