Immunoassays For The Detection Of Four Mycotoxins In Maize

Mycotoxins are natural metabolites,produced by fungi.They can affect countless varieties of food such as maize and wheat,and provide high toxicology which threatens human and animals' health.The immunoassay with highly sensitive,rapid and high throughput is an important method to avoid human and animals suffering from mycotoxins.Antibody is a key factor for developing immunoassay;double-wavelength fluorescence polarization immunoassay(FPIA)is an effective way for improving the high throughput;new environment friendly namomaterials as labels used in the lateral flow immunoassay(LFA)is an important stragety for enhancing the sensitivity.The aim of this study is to prepare broad-specific and highly sensitive monoclonal antibodies(mAbs)and establish a double-wavelength FPIA and a multi-LFA for screening multiple mycotoxins in maize.The technical bottleneck of the class-specific antibodies with highly sensitivity and uniform cross-reactivity simultaneously was broken by using a variety of low-affinity competitor for screening the hybridoma cell supernatant.Two class-specific mAbs against aflatoxin(AFs)and zearalenones(ZENs),named 5H3 and 3D4,were produced.The half maximal inhibitory concentrations(IC50)of mAb 5H3 against aflatoxin B1(AFB1)was 0.0928 ng/mL using direct competitive ELISA,and the cross-reactivity(CR)were 80.1%,133.7%,81.6%,94.2%and 60.9%towards aflatoxin B2(AFB2),aflatoxin G1(AFG1),aflatoxin G2(AFG2),aflatoxin M1(AFM1)and aflatoxin M2(AFM2),respectively.IC50 of mAb 3D4 against ZEN was 0.041 ng/mL using indirect competitive ELISA,and the CR were 66%,107.9%,57.2%,103.5%and 73.9%towards zearalanone(ZAN),a-zearalenone,(a-ZOL),?-zearalenone(?-ZOL),a-zearalanone(a-ZAL),?-zearalanone(?-ZAL),respectively.In addition,a highly sensitive mAb 12E8 against deoxynivalenol(DON)with IC50 value of 2.95 ng/mL and a highly specific mAb 9C7 against T-2 toxin(T-2)with IC50 value of 1.12 ng/mL were prepared.The CR of the 12E8 towards 3-acetyl-deoxynivalenol(3-Ac-DON)and 15-acetyl-deoxynivalenol(15-Ac-DON)were 2681%and 23%,respectively,while mAb 9C7 did not exhibit measurable CR with other mycotoxins.A homogeneous double-wavelength fluorescence polarization immunoassay(DWFPIA)for simultaneous detection of AFs and ZENs in maize based on the spectral resolution technology was developed.Under optimum conditions,the pairs of AFB1-EDF-mAb 5H3 and ZAN-TRCA-mAb 3D4 was selected as the best pairs of fluoresicein tracers-antibodies..The IC50 values against the AFs cocktail standard solution(AFB1,AFB2,AFG1 and AFG2 weight ratios of 1:1:1:1)and ZENs cocktail standard solution(ZEN,ZAN,a-ZAL,?-ZAL,?-ZOL and ?-ZOL weight ratios of 1:1:1:1:1:1)were 2.68 ng/mL and 4.08 ng/mL,and the responding limit of detection in maize samples were 4.98 ?g/kg and 11.03 ?g/kg,respectively.Recoveries ranged from 78.6%to 103.6%with coefficients of variation(CV)below 19.2%.We provided a simple preparation procedure for amorphous carbon nanoparticles(ACNPs)and described multiplex LFAs employing ACNPs as labels(ACNPs-LFA)for detecting three Fusarium mycotoxins,DON,T-2 and ZEN.Under optimized conditions,the visual limit of detection(vLOD)of ACNP-LFAs in maize were 300 ?g/kg for DON,150 ?g/kg for T-2 and 15 ?g/kg for ZEN,while the quantitative limit of detection(qLOD)were 19.8 ?g/kg for DON,12.9 ?g/kg for T-2 and 1.04 ?g/kg for ZEN.The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109%and CV below 13%.The results of ACNPs-LFA using naturally incurred maize samples showed good agreement with results from HPLC-MS/MS.In a word,the double-wavelength FPIA and multi ACNPs-LFA based on the broad-specific and highly sensitive mAbs could meet the requirement of high throughput and simultaneous on-site screening of multiple mycotoxins in maize.