Studies On Surrogate Based Green Immunoassay For Aflatoxin

The aflatoxins are mycotoxins produced mainly by the fungal species Aspergillus flavus，Aspergillus parasiticus and Aspergillus nomius, which occur on a wide range of agriculturalcommodities and food matrices, such as peanuts, corns, soybeans, rice and sorghum, etc. Aflatoxin is upto now one of the most potent carcinogens, which is harmful to human and livestock. Every country inthe world has set up strict regulations towards aflatoxins in food and agro-products. Some developedcountries set up trade barrier on aflatoxins which cause serious economic loss to our export ofagro-products due to exceeding concentration of aflatoixns. Mycotoxin-producing fungi can occur inany food chain and will produce aflatoxins under some circumstances. As a result, the occurrence ofaflatoxin is unavoidable and difficult to be elimited by regulation. In order to protect the health ofcustomers, eliminate the economic loss, guarantee the sustainable development of society and realizethe Chinese Dream of ‘eat safe’, powerful detection techniques are required to avoid harmful foodentering into the market and onto the table.There are a variety of analytical methods for aflatoxins, including the original thin-layerchromatographic (TLC) method, immunoaffinity column purification coupled with fluorescencedetection, high performance liquid chromatography (HPLC), etc. Immunoassays are importantsupplement to those methods, which have advantages such as high sensitivity, selectivity, rapid samplepreparation, and cost effective, etc. Aflatoxins are small moleculars, so it is necessary to use anaflatoxin-protein conjugate as coating antigen, competing combination to antibody with free aflatoxins,so called competitive immunoassay. In the case of aflatoxins, the coating antigen is always synthesizedby conjugating protein, such as bovine serum albumin to aflatoxin molecular. Huge amount of aflatoxinstandard is needed during synthesis, as well as in the development of an immunoassay, which is notonly quite expensive, but also causes potential hazardous to analysts and the environment.In our research, two types of aflatoxin substitute antigen and one surrogate standard were obtained.Green immunoassays based on surrogate antigen or standard were developed in this research, whichprovide a sensitive, fast, simple, cost effective and environmental friendly method to regulate aflatoxinsin agro-products. The main contents and innovations are as follow:1. Phage-displayed peptides that mimic aflatoxin antigen were developed in this research. Greenimmunoassay towards aflatoxin was constructed based on those mimotopes, which provide technicalsupport to green analysis of aflatoxin.A random-8-peptide phage-displayed library was constructed and used as a source of peptides thatmimic aflatoxins. Five mimotope peptides were obtained by panning-elution from the library. Afteroptimization of the concentration of coating antibody and phage, pH value, ion strength, and methanolconcentration, etc, competitive ELISAs were constructed based on five phage-displayed peptides.ELISA based on phage CM4has the highest sensitivity with IC50value of0.290ng/mL. Matrix effect was studied by using peanut, corn and rice as representative samples. By adding2-4%BSA in extractdilution buffer, matrix effect was successfully eliminated to an acceptable range. Further validationindicated relatively good recovery (60-120%) with RSD less than10%. Natural contaminated sampleswere analyzed for aflatoxin concentration by both conventional ELISA and phage ELISA. The resultsshowed good correlation (R2=0.931). It can be concluded that the mimotope preparation is an effectivesubstitute for the aflatoxin based coating antigen in ELISA and can be used in real sample analysis.2. This is the first application of nanobody technique in the development of antigen mimotope,which provides a new technical platform for green analysis of aflatoxin.An alpaca was immunized by anti-aflatoxin monoclonal antibody1C11. Total RNA was extractedfrom alpaca’s blood and VHH genes (400-600bp) were cloned by PCR using two pairs of hingespecified primers. The phage displayed VHH library was constructed by ligating amplified VHH geneswith plasmid pComb3X and electrotransformated to E. coli ER2738. The achieved library has a size of1.1109pfu. After3rounds of biopanning, three anti-idiotype nanobodies were selected. Phage ELISAwas developed after a series of optimization of coating antibody concentration, phage workingconcentration, pH value, ionic strength and methanol concentration, etc. ELISA based on phage2-5hasthe best sensitivity with IC50value of0.054ng/mL,0.140ng/mL,0.077ng/mL and0.373ng/mLtowards AFB1, AFB2, AFG1and AFG2, respectively. Three anti-idiotype nanobodies were expressed,purified and used directly as substitute coating antigen. After optimization of coating concentration,antibody concentration, pH value, ionic strength and methanol concentration, VHH ELISAs weredeveloped with three nanobodies. The best immunoassay developed with VHH2-5shows an IC50of0.160ng/mL towards aflatoxin B1and cross-reactivity toward aflatoxin B2, G1and G2of54.4%,90.4%,and37.7%, respectively. Good recoveries (70-120%) of aflatoxin B1were achieved from peanuts, riceand corn. A good correlation was found between the data obtained from the conventional ELISA andELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. Theseresults indicated that the developed ELISA could well meet the specific detection of AFB1in realsamples.3. This is the first research using nanobody as aflatoxin surrogate standard, which exploits a newway to develop immunoreagent.Nanobody VHH2-5was applied as surrogate standard in competitive ELISA towards aflatoxin. Byusing the concentration of VHH2-5as x axis, the value of B/B0as y axis, a standard curve wasdeveloped. The concentration of VHH2-5showed excellent linear relationship with aflatoxinconcentration at the same inhibition value (R2=0.998). The concentration of aflatoxin was calculated bya two-step calculation: the concentration of VHH2-5was first achieved by a four-parameter logisticregression from the detected OD450value, and then converted to aflatoxin concentration by a linearequation. The average recoveries of AFB1from spiked peanut, rice and corn samples were in the rangeof70-110%. The inter-and intra-laboratory reproducibility was in the range of0.7-6.2%and8.3-18.7%, respectively. The assay was compared to a high-performance liquid chromatographic (HPLC) methodfor determination of20naturally contaminated peanut samples, displaying a good correlation(R2=0.988). We also tested the thermostability of VHH2-5, by comparison with anti-idiotypepolyclonal antibody towards aflatoxin M1developed in our laboratory, VHH2-5showed better stabilityunder high temperature. In conclusion, VHH2-5could be used as surrogate standard and applied in thecontamination monitoring of aflatoxins in real samples.