Fluorescent Immunochromatographic Assay For Quantitative Determination Of Mycotoxins In Grain

The life and health of humans and animals are seriously threatened by food safety issues,including microbial contamination,agricultural drugs and veterinary drugs residues,and the addition of prohibited substances.Foodborne diseases induced by mycotoxins produced by microorganisms in foods have attracted widespread attention.More than 300 compounds are now recognized as mycotoxins,of which approximately30 are more harmful.Especially aflatoxin B₁（AFB₁）,zearalenone（ZEN）and ochratoxin A（OTA）,have the highest probability of pollution.They are mainly found in food and its products.And some fungi can produce a variety of toxins at the same time,and it is also common for a variety of toxins to simultaneously contaminate a grain.Strengthening the research and development of testing technology is an important means to ensure food quality and safety.Fluorescent immunochromatographic assay（FICA）has the advantages of sensitivity,accuracy,rapidity,high throughput,low cost,etc.There are a variety of fluorescent markers to choose from,and multiple components can be detected simultaneously.This paper aims to establish a fluorescent immunochromatographic assay for the detection of mycotoxins in food,providing reference for government regulation.First,the hapten AFB₁-CMO was obtained by sputum modification of AFB₁,and the carrier protein keyhole limpet hemocyanin（KLH）,bovine serum albumin（BSA）and egg serum albumin（OVA）were coupled by active lipid method,synthetic whole antigens（AFB₁-CMO-KLH,AFB₁-CMO-BSA and AFB₁-CMO-OVA）.Then,a cell strain capable of stably secreting AFB₁ monoclonal antibody was prepared by animal immunization and monoclonal antibody technology,and named as 3B6,the heavy chain was IgG1 type,the light chain wasλtype,and half maximal inhibitory concentration(IC₅₀)was 0.01112μg/L,the cross-reaction rate of other structural analogs were less than 22.8%,and the cross-reaction rate of other mycotoxins,antibiotics and agonists was less than 0.1%.Establishing three kinds of fluorescence immunochromatographic assays（TRFN-FICA,QB-FICA,and QD-FICA）for detecting AFB₁ in grain based on time-resolved fluorescent microspheres（TRFN）,quantum dot microspheres（QB）,and quantum dots（QD）.For the first time,the three methods were systematically and comprehensively compared using the same antigen and antibody.The results showed that TRFN-FICA had less coating antigen consumption（0.30μg,0.65μg and 0.65μg）,less antibody was used in each test strip（0.015μg,0.09μg and 0.03μg）,and shorter immunoassay time（25minutes,30 minutes and 35 minutes）than QB-FICA,and QD-FICA.Under optimal conditions,the detection limits of TRFN-FICA,QB-FICA and QD-FICA were 0.00061ng/mL,0.00405 ng/mL and 0.01066 ng/mL,with the quantitative detection ranges of0.00368⁰.04804 ng/mL,0.00921⁰.06375 ng/mL and 0.0156⁰.07776 ng/mL,respectively.TRFN-FICA has the highest sensitivity and the widest detection range.The recovery of TRFN-FICA ranged from 83.64%to 125.61%,and the coefficient of variation ranged from 3.10%to 6.75%;the recovery of QD-FICA ranged from 80.29%to 129.45%,and the coefficient of variation ranged from 2.88%to 7.16%.The recovery of QD-FICA ranged from 64.53%to 133.86%,and the coefficient of variation ranged from 2.34%to8.96%.The accuracy and precision of TRFN-FICA are optimal.Simultaneous analysis of60 food samples by liquid chromatography tandem mass spectrometry（LC-MS/MS）,TRFN-FICA,QB-FICA and QD-FICA,TRFN-FICA have the highest consistency with the LC-MS/MS.Compared to QB-FICA and QD-FICA,TRFN-FICA is more suitable for quantitative detection of aflatoxin B₁ in grain.A multiple time-resolved fluorescent microsphere immunochromatographic assay for AFB₁,ZEN and OTA in grain was established.Combined with a portable multi-channel fluorescence reader,this method can simultaneously obtain quantitative detection results of three toxins within 15 mim.Under optimal conditions,the IC₅₀ of the method for AFB₁,ZEN and OTA were 0.01638 ng/mL,0.06427 ng/mL and 0.04496 ng/mL,respectively.The quantitative detection range was 0.00387-0.06924 ng/mL and 0.01435-0.28789 ng/mL and 0.0099-0.20423 ng/mL.Three toxins were added separately or in combination with LOD,2LOD,and 4LOD in six food samples（corn,soybean,sorghum,wheat,rice,oats）.The recovery rates of AFB₁,ZEN and OTA in the six food samples were 66.94%-148.41%,71.38%-145.29%and 68.94%-138.00%,respectively.The CV were 2.38%-6.91%,3.04%-7.3%and 2.38%-7.78%,respectively.The detection value of the actual sample is in good agreement with the detection result of LC-MS/MS.The method has broad application prospects in the field of mycotoxin mixed pollution monitoring.