Study On Chemiluminescence Magnetic Enzyme Immunoassay Of Zearalenone And Ochratoxin A

The safety problems caused by mycotoxins have become the focus of attention all over the world.At present,more than 500 kinds of mycotoxins have been found.Among them,zearalenone(ZEN)and ochratoxin A(OTA)widely exist in food.They have the characteristics of strong toxicity such as carcinogenicity and teratogenicity,and seriously endanger the health of human and animals.At present,chromatographic analysis technology has developed into a standard method for the determination of mycotoxins,including high performance liquid chromatography and high performance liquid chromatography tandem mass spectrometry.These detection methods have the advantages of high sensitivity and good accuracy.However,these methods usually need large-scale experimental equipment,complex sample pretreatment,cumbersome operation process and skilled professional operators,this greatly limits the detection efficiency.The commonly used rapid detection methods of mycotoxins are enzyme-linked immunosorbent assay(ELISA)and colloidal gold chromatography.The former is cumbersome and time-consuming and requires high professionalism of testers.Although the latter is simple,the detection sensitivity is low,and the samples to be tested need to be tested one by one,which can not be automated,resulting in low detection efficiency.Based on the principle of indirect competition,taking ZEN and OTA as the research object,superparamagnetic magnetic beads as the solid-phase carrier and combined with high-sensitivity chemiluminescence technology,a chemiluminescence magnetoenzyme immunoassay(MECLIA)was established for the low-cost,sensitive and rapid quantitative detection of ZEN and OTA in grain.The main research contents and results are as follows:1.Establishment and application of zearalenone chemiluminescence assayStreptavidin magnetic beads(SA-MB)was used instead of traditional micropores as solid carrier to realize three-dimensional reaction,shorten immune reaction time and improve detection sensitivity;Goat anti mouse IgG(H+L)labeled with alkaline phosphatase(ALP)was used as enzyme labeled secondary antibody to generate detection signal;ZEN monoclonal antibody is used as the primary antibody,which can connect ZEN and enzyme labeled secondary antibody;Alkaline phosphatase luminescent substrate 01(APS)was used as substrate;The sample to be tested competes with ZEN biotin to bind the primary antibody.After washing,the amount of enzyme labeled secondary antibody on the surface of SA-MB is inversely proportional to the amount of substance to be tested in the sample,that is,the detection signal intensity is inversely proportional to the amount of substance to be tested in the sample.The dilution ratio of primary antibody and ZEN biotin was determined by checkerboard method,and the key parameters such as sample extraction time were optimized.The correlation between ZEN concentration and chemiluminescence signal intensity was calculated by four parameter fitting,and the working curve was established;When detecting ZEN in corn,wheat,brown rice,flour and other samples,the linear range of the method is 30～180 μg/kg;the detection limit in corn matrix was 4.03μg/kg;In this method,ZEN has no obvious cross reaction with common mycotoxins aflatoxin(B1,B2,M1,M2),T-2 toxin,ochratoxin A,acetyldeoxynivalenol,nivalenol,etc;The recoveries of low,middle and high levels were 109.5%,87.0%and 101.8%,and the relative standard deviation was 8.1%;The detection results of this method are in good agreement with those of HPLC-MS.the quantitative detection of ZEN in 20 samples can be completed in 25 minutes.In conclusion,the results show that this method has the advantages of high sensitivity,good specificity,high accuracy and good repeatability,which provides a technical means for the rapid quantitative analysis of ZEN.2.Establishment and application of chemiluminescence magnetic enzyme immunoassay for ochratoxin ASA-MB was used as the solid carrier of immune reaction;ALP labeled Goat anti mouse IgG(H+L)was used as enzyme labeled secondary antibody;Using OTA monoclonal antibody as primary antibody and APS as substrate,OTA chemiluminescence magnetic enzyme immunoassay was established.Firstly,the extraction conditions of anti OTA and the proportion of anti OTA were optimized.The linear range of this method for the determination of OTA in corn,wheat,brown rice,flour and other samples is 2.5～10 μg/kg,the detection limit in corn matrix was 2.29μg/kg;In this method,OTA has no obvious cross reaction with common mycotoxins aflatoxin(B1,B2,M1,M2),T-2 toxin,zearalenone,acetyldeoxynivalenol,nivalenol,etc,and has good specificity;The recoveries of low,middle and high levels were 96.3%,103.8%and 92.2%,and the relative standard deviation was 6.0%.Therefore,the detection precision,specificity,sensitivity and linear range of OTA chemiluminescence magnetic enzyme immunoassay established in this study meet the requirements,and can realize the rapid quantitative detection of 20 samples within 25 minutes,which lays a good foundation for the automatic detection of large quantities of samples.The incubation and washing process of chemiluminescence magnetic enzyme immunoassay established in this study is automated with the help of mycotoxin automatic purifier,which can significantly reduce error,improve the repeatability of detection results,and realize the high sensitivity,low cost and easy automation of trace mycotoxins in complex matrix.It is of great significance and application value to ensure food safety and consumer health.