Development Of Nanobody Against Aflatoxin And Studies On Its Immunoassay Technology

Aflatoxins are one of the strongest chemical carcinogens yet found.They commonly contaminate agricultural commodities and food matrices,such as maize,peanut,rice and sorghum,which exhibits great health endangering and life threatening to human and animals.Exported agro-products of China are continuously detained,returned or even destructed due to the exceeding aflatoxin content,which bring enormous economic loss to China.Aflatoxin contamination has become a potential risk in agro-products quality and food safety field.Apart from the biocontrol measures to toxigenic fungus（e.g.Aspergillus flavu,Aspergillus parasiti,etc.）,highly effective and sensitive analysis methods towards aflatoxins in post-harvest of agro-products are urgently required,to prevent harmful food entering into the market and onto the table,thus ensuring the consumption safety of agro-products and people’s health.Currently,the main method of Aflatoxin detection includes physicochemical analysis based on chromatography（e.g.TLC,HPLC,LC-MS/MS,etc.）and immunoassay on the basis of specific binding of antibody to antigen（e.g.enzyme-linked immunoassay,immunochromatography assay,immunosensor,etc.）.Extroadinarily,enzyme-linked immunoassay（ELISA）-based immunoassays has the merits of high sensitivity and good selectivity,in which the preparation of high-quality antibodies is the key.The development of antibody has experienced polyclonal antibodies,monoclonal antibodies and genetic engineering antibodies.Compared with conventional polyclonal antibodies and monoclonal antibodies,genetic engineering antibodies possess the advantages of high yield in bacteria strain in short time,cost effective,stable genetic information and facile manipulation of antibody affinity,which introduce them into the field of food safety and environmental pollutants detection in recent years.This work based on single chain antibody against aflatoxin,anti-idiotypic F（ab’）₂ antibody and anti-idiotypic nanobody prepared in our previous research.From immunized alpaca,we constructed a phage-displayed VHH library and selected variable domain of heavy chain of heavy chain antibody（VHH）against aflatoxin,termed VHH antibody or nanobody.ELISA based on phage-displayed VHH was developed for aflatoxin detection.We also developed soluble VHH-based ELISA,VHH-based immunoaffinity column and VHH-based lateral flow time-resolved fluorescence immunoassay（TRFIA）.These established methods provide a new direction for genetic engineering antibody production and application in aflatoxin immunoassay.The main contents and innovations of this research are as follows:1.Phage-displayed VHH Library was first constructed in this research and phage-displayed VHH based ELISA was developed to aflatoxin B₁ detection in agro-products.An alpaca was immunized with AFB₁-BSA.Total RNA was extracted from alpaca’s peripheral blood lymphocytes and reverse transcribed into cDNA.The VHH gene was cloned by PCR and ligated with phagemid vector pComb3X and then electrotransformed into E.coli ER2738.A phage-displayed VHH library was achieved with a size of 2×10⁷ pfu.After 4 round of biopanning,two anti-aflatoxin B₁nanobodies were selected,termed Nb26 and Nb28.Phage-displayed VHH based ELISA（phage-ELISA）was developed after a series of optimization of coating antigen concentration,blocking reagents,pH value,ionic strength and methanol concentration,etc.ELISA based on phage-Nb26 and phage-Nb28showed the best sensitivity with IC₅₀ value of 0.317ng/mL and 1.191 ng/mL,respectively.Phage-ELISA based on Nb26 was developed and good recoveries（81.0¹12.0%）of aflatoxin B₁ were achieved from peanuts,rice,corn and feedstuff.Consensus results were obtained from the phage-ELISA and HPLC in analysis of aflatoxin B₁ contaminated in samples.These results indicated that the developed phage-ELISA could be applied to AFB₁ contamination monitoring,and provide technical support for building phage-displayed VHH library and select specific nanobodies towards other mycotoxins.2.The soluble nanobody without phage was prepared and its stability was investigated.The development of nanobody based ELISA（VHH-ELISA）improve the possibility for the application of genetic engineering antibody in immunoassay.In order to prepare the nanobody without phage,an orthogonal design was carried out to optimize the expression conditions in bacteria,which result in about 30%increase of yield for soluble nanobody.The thermostability of nanobody was investigated and compared with monoclonal antibody mAb B5against AFB₁.The results show that the nanobody has better tolerance in heat,acid environment,high ionic strength and high concentration of organic solvent than that of monoclonal antibody.After a series of optimization of nanobody based ELISA（VHH-ELISA）,ELISA based on nanobody Nb26 and Nb28have the best sensitivity with IC₅₀ value of 0.754 ng/mL and 1.012 ng/mL,respectively.Take advantage of high tolerance to methanol of nanobody,inhibition curve of nanobody Nb26 towards AFB₁ was established under the condition of 70%methanol and compared with the sample matrix curve when peanut,rice,corn and feedstuff extracted by 70%methanol solution.The resultant curves show little significant difference and the sensitivity in real sample detection of Nb26 is 3.45μg/kg,linear range is2.47²68.6μg/kg,AFB₁ recovery rate in real sample is 86.3¹12.0%.These results suggest that the established VHH-ELISA method can be applied into aflatoxin contamination monitoring.3.We firstly use nanobody as adsorbent in immunoaffinity column that can be used repeatedly.The pretreatment of agro-products can be purified by regenerated nanobody based immunoaffinity column.Preparation of nanobody Nb26 based immunoaffinity column and compared with monoclonal antibody mAb 1C11 and mAb 10C9 based immunoaffinity column.The VHH-IAC showed better dissociation ability toward AFB₁ than that of mAb-IAC.Recovery rate of VHH-IAC with reusd 10times was over 80%,while the recovery rate of mAb-IAC was decreased rapidly as reuse times increased.VHH-IAC with reused 15 times was applied to pretreat real sample and compared with the first use of the commercial mAb-IAC.A good correlation was found between the detection results obtained from these two kinds of immunoaffinity column.These results suggest that VHH-IAC can be used repeatedly as pretreatment tool when AFB₁ content detected by large-scale instrument analysis.4.A lateral flow time resolution fluorescent immunoassay（TRFIA）based on nanobody was first established,which provides a new technical platform for genetic engineering antibody in rapid analysis of mycotoxins.A novel TRFIA was developed for quantitative detection of AFB₁,by utilizing nanobody-europium labeled polystyrene nanospheres conjugates as fluorescence probes.After optimization of the conjunction amount of nanobody,buffer,the amount of antigen on test line and second antibody on control line,dilution rate of the fluorescence probes,reaction time and concentration of methonal,nanobody-based TRFIA was performed for AFB₁ detection and five standard curves were established in buffer,peanut,rice,maize and feed.The linear range of this method for buffer is 0.1⁵0μg/kg,LOD is0.05μg/kg.For four different matrixs,the linear range is 0.2⁴0,1.0⁵0,1.0⁴0,0.5⁴0μg/kg,the LOD is 0.06,0.34,0.25,0.17μg/kg for corn,peanut,rice and feed,respectively.Recovery rate in real sample by VHH-TRFIA is 78.1%⁹2.6%.A good correlation was found between the detection results obtained from the VHH-TRFIA and HPLC in analysis of aflatoxin B₁ contaminated in samples.This established VHH-TRFIA method providing a new way for rapid detection of mycotoxins in food safety,the research and application of nanobody is expected to be carried out further more.