Studies On Anti-idiotypic Nanobody Based Green Immunoassay For Zearalenone

Zearalenone(ZEN)is produced by several Fusarium species wich mainly contaminates grains including corn,wheat,sorghum.ZEN wich has significant estrogenic-like effect can cause reproduction toxicity,immunity toxicity,liver toxicity,genotoxic activity.ZEN can threaten human health and the development of animal husbandry by contmainating cereals and their products,milk,meet and so on.Owing to the level of ZEN contamination in food commodities and dietary exposure,many countries have set the maximum level of ZEN in foodsupplements.At present,there are a diversity of analysis methods for ZEN,including thin layer chromatography,high-performance liquid chromatography(HPLC),immunoassays and electrochemical sensor,etc.By contrast,immunoassay methods are specific to analytes based on antibody-antigen interactions,which also have advantages such as high sensitivity,rapid sample preparation,fast detection and cost effective,etc.Thus,immunoassays are suitable for rapid screening of a large number of samples.However,huge amount of ZEN standard and organic reagents are needed for synthesis of chemically-derived detection antigen,as well as in the development of an immunoassay,which causes potential hazardous to analysts and the environment.In this present study,we choose model small molecules mycotoxin,zearalenone(ZEN)as an analyte.The anti-idiotypic nanobody technology was applied for ZEN molecular mimicry research.ZEN substitute antigens based on nanobody were obtained,and ZEN green immunoassays based on surrogate antigen or standard were developed in this research,which improved operator safety by reducing the risk of exposure to toxic reagents.The study provides a sensitive,fast,simple,cost effective and environmental friendly method to monitor ZEN contamination.1.This is the first straightforward acquisition of AIds from an alpaca immunized nanobody library and application of nanobody technique in mimicry of ZEN antigen.The green immunoassays based on nanobody are developed,which provide a new tool for other toxic small molecules detection.An alpaca was immunized by anti-zearalenone monoclonal antibody 2D3.Total RNA was extracted from alpaca's blood and VHH genes(400-600 bp)were cloned by PCR using two pairs of hinge specified primers.The phage displayed VHH library was constructed by ligating amplified VHH genes with plasmid pComb3X and electrotransformated to E.coli ER2738.The achieved library has a size of 4.6×107 pfu.After 4 rounds of biopanning,one anti-idiotypic nanobody was selected.Phage ELISA was developed after a series of optimization of coating antibody concentration,phage working concentration,pH value,ionic strength and methanol concentration,etc.ELISA based on phage 8#has high sensitivity with IC50 value of 0.207 ng/mL towards ZEN.The anti-idiotypic nanobody was 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 were developed with this nanobody.The immunoassay developed with VHH-8#shows an IC50 of 0.118 ng/mL towards zearalenone.Good recoveries(71.7%?102.2%)of zearalenone were achieved from corn,wheat,and feedstuffs.2.This is the first research using nanobody as zearalenone surrogate standard,which exploits a new way to develop immunoreagent.Nanobody VHH-8#was applied as surrogate standard in competitive ELISA towards zearalenone.By using the concentration of VHH-8#as x axis,the value of B/B0 as y axis,a standard curve was developed.The concentration of VHH-8#showed excellent linear relationship with zearalenone concentration at the same inhibition value(R2=0.984).The concentration of zearalenone was calculated by a two-step calculation:the concentration of VHH-8#was first achieved by a four-parameter logistic regression from the detected OD450 value,and then converted to zearalenone concentration by a linear equation.The average recoveries of ZEN from spiked corn,wheat,and feedstuffs samples were in the range of 80.8%?101.3%.We also tested the thermostability of VHH-8#,and VHH-8#showed very good stability under high temperature.In conclusion,VHH-8#could be used as surrogate standard and applied in the contamination monitoring of zearalenone in real samples.