Study On Improvement Of Sensitivity Of Anti-idiotypic Nanobody Based Immunoassay For Citrinin

Citrinin,a type of secondary fungal metabolite,is produced by several species of the general Penicillium,Aspergillus and Monascus.Citrinin contamination has been widely existed in agricultural products,feedstuff and monascus products.As a mycotoxin,citrinin is not only an antibiotic,bacteriostatic,antifungal,and antiprotozoal substance but is also a known hepato-nephrotoxin to a wide range of species.In addition,it has been experimentally proved that citrinin possess potential genotoxicity,tumorigenicity,carcinogenicity,embryotoxicity,and teratogenicity.Owing to the negative health effects and potential citrinin contamination in various food products,several Food Safety Agencies worldwide have made respective maximum residue limits.At present,the quantitative detection method of citrinin mainly includes: TLC,HPLC,biosensor,immunoassays.Immunoassays can be used to quantitative determination of target molecule based on the specific binding of antigen and antibody,compared with other analytical approach,it has the characteristics of high sensitivity,good specificity and simple pre-processing.Nevertheless,immunoassays are easy to be affected by matrix effects which has the negative effect on reliability of detection results.Improving the sensitivity of immunoassays is an effective way to lower the matrix effects of complex samples,therefore,two strategy have been used to impove the sensitivity of mimotope A9-based immunoassay in this study.The main contents are as follows:1.Development of real-time immuno-PCR method for quantitative determination of CITThe mimotope A9-based real-time immuno-PCR method had been established after optimal experiments of annealing temperature and amplification efficiency of q PCR,concentration of coating antibody,A9 phage and methyl alcohol.The IC50 value of established method in present study is 9.86±2.52 ng/m L which is nearly equivalent to the traditional phage-ELISA method.However,the linearity range of established method is 0.1-1000 ng/m L which had been 10-fold broaden than phage-ELISA method.Besides,the mimotope A9-based real-time immuno-PCR method has no crossreactivity to the common mycotoxins,like AFB1,DON,OTA and ZEN.The method has also been appied to the determination of CIT in wheat and rice samples,the recovery was founded to be in the range of 90.0%-104.6% and 75.8%-110.0% respectively.There was no significant difference in results between the method of realtime immuno-PCR and UPLC-MS.2.Confirmation the key sites by means of computer molecule simulationIn this study,the three-dimensional structure of mimotope A9 and CIT Sc Fv had been built through “homologous modeling” strategy.Then,CIT and A9 had been respectively docked to CIT Sc Fv through “molecular docking” program.Finally,T28,F29,N30,R31 and Y32 had been confirmed as the key binding sites in A9.When all these five amino acid had been mutated to “alanine”,results of phage-ELISA showed that mutational phage had lost the binding activity to CIT Sc Fv,which proves the correctness of molecule simulation.3.Generation and screen of site-directed saturation mutants“Site-directed saturation” strategy had been used to produce mutants at position 28-32 and 74,an average of 18 different amino acids had been obtained at those position.Then,the activity of all the mutants had been identified by indirect-competive phage-ELISA.Mutants T28F?T28I?F29I?F29V?N30T?N30V had been demonstrated have higher sensitivity than the wide-type A9 by 1.83,1.37,1.70,2.96,1.31 and 2.01-folds,respectively.