Application Of Aflatoxins Mimetic Peptide And Its Structural Analogues In IAC-HPLC Detection

Aflatoxins（AFT）are a kind of mycotoxin that seriously endangers food safety and has extremely strong carcinogenicity,teratogenicity,and mutagenicity.Immunoaffinity column-high performance liquid chromatography is a classical method for the detection of AFT with high sensitivity and precision.As an important pretreatment method,the Immunoaffinity column（IAC）is widely used for the detection of AFT.However,the IAC has a problem that the real-time monitoring of the column capacity is difficult.Therefore,this study started from two directions.First,by using phage display technology,we selected the mimetic peptides that could bind to AFT antibodies.Second,performing molecular chemical modification on AFT to obtain an AFT modifier;Through the study of these two substances,a real-time monitoring method for the capacity of AFT immunoaffinity column was explored,which has a great significance for the IAC combined instrument detection method.The main research contents are as follows:1.Screening of the aflatoxin mimetic epitopesUsing the anti-AFB₁2E10 monoclonal antibody as the target molecule,a phage display random cyclic heptapeptide peptide library was used to screen the phage minic epitopes.After four rounds of screening,a total of 37 phage clones were selected,and 32 positive phage clones with specific binding were identified by the ELISA method,of which 23 positive clones were effectively inhibited by AFB₁.DNA sequence analysis showed that these 23 phage particles were six different DNA sequences.Clone A1 and Clone A5 had a common sequence,the common sequence was isoleucine（I）-proline（P）-tryptophan（W）.The competition ELISA curves were established to test the six phage minic epitopes,and the linear range of those curves were around 0.0125-25 ng/m L with similar IC₅₀values.2.Synthesis and verification of mimetic peptideThe DNA sequence of B₁with the most enriched amount was selected for chemical synthesis mimetic peptide.In the indirect competitive ELISA of the mimetic peptide,the optimal reaction system was as follows:artificial antigen coating concentration was 0.5μg/m L,antibody dilution ratio was 1:16000,sample dilution was 20%methanol-PBS solution,competitive binding time was 5 min,and ion strength was 100 m M.The linear range of the ELISA curve was 2.5～500μg/m L,and the IC50 value was 25μg/m L with a good linear relationship.Furthermore,an ELISA method using mimetic peptide-BSA as a coating antigen was established.The optimal coating concentration was 50μg/m L,and the optimal antibody dilution was1:400.From the IC50 values of the mimetic peptide and the working condition of the peptide-BSA conjugate,it was found that the affinity of the synthetic peptide to the antibody was much lower than that of AFB₁to the antibody,which indicated that the synthetic peptide could not be used for real-time monitoring of column capacity.3.Study on chemical synthesis,purification and identification of AFT structuralmodification productsTwo structural analogues of AFT（AFB₁and AFB₂）were proximate with CMO in pyridine,respectively,to obtain the modification products of AFB₁and AFB₂（（Z）-2-（（（4-methoxy-11-oxo-3,6a,9a,11-tetraphydrocyclopenta[c]furo[3’,2’,:4,5]furo[2,3-h]chromen-1（2H）-ylidene）amino）oxy）acetic acid and（Z）-2-（（（4-methoxy-11-oxo-3,6a,8,9,9a,11-hexaphydrocyclopenta[c]furo[3’,2’,:4,5]furo[2,3-h]chromen-1（2H）-ylidene）amino）oxy）acetic acid,named AFB₁-D₁and AFB₂-D₁）.The study showed that the aflatoxin M₁（AFM₁）immunoaffinity column could bind the two products.The fluorescence and ultraviolet spectra showed that the excitation and emission wavelengths of AFB₁-D₁and AFB₂-D₁were similar to those of AFM₁,and the ultraviolet absorption wavelength ranges were relatively consistent.The relative molecular masses determined by mass spectra were consistent with expecting.The standard curve of competitive ELISA of AFB₁-D₁was established,and the linear equation was obtained as follows:y=-0.5213x+0.9915,R²=0.9954,IC50=8.45ng/m L,concentration range 0.1562～25.125 ng/m L,and the cross-reactivity ratio of AFB₁-D₁to AFM₁was 5.14%.The AFB₂-D₁competitive ELISA was established within the concentration range of 4.3945～2250.125 ng/m L,with the linear equation of Y=-0.3072x+0.118,R₂=0.9970,IC50=105.17 ng/m L,and the cross-reactivity ratio of AFM₁-D₂to AFM₁standard was 0.37%.4.Application of AFT structural modifier in IAC-HPLCIt was confirmed by the spiked recovery experiment that the recoveries of AFB₁-D₁and AFB₂-D₁in the AFM₁immunoaffinity column were greater than 80%and less than 60%,respectively.AFB₁-D₁could be used for real-time monitoring of column capacity in the next research.For an AFM₁immunoaffinity column with a column capacity of 100 ng,AFB₁-D₁additions of 50 ng,75 ng,and 100 ng were set,with AFB₁-D₁addition of 0-100 ng;The experimental results showed that when the dosage of AFB₁-D₁was 100 ng,the average recovery of AFM₁was within the range of 84.1%～93.2%and the relative standard deviation was within the range of0.97%～2.79%.The mean recovery of AFB₁-D₁was 43.2%～82.3%and the relative standard deviation was 0.88%～5.69%.The total binding amount ranged from91.22～127.39 ng.The detection accuracy and repeatability are good,and the column capacity monitoring can be realized.The addition level of AFB₁-D₁was chosen to be100 ng,and the spiked recoveries in different matrices were tested.The results showed that the average recovery ranges of AFM₁in milk powder and pure milk were 86.0%～95.6%and 90.69%～101.6%,respectively.The relative standard deviations（RSD,n=3）were 1.0%～5.21%and 1.35%～4.16%,respectively.The average recoveries of AFM₁-D₁were 46.14%～81.71%and 42.6%～92.08%,respectively,with relative standard deviations（RSDs）in the range of 1.13%～6.24%and 0.49%～1.03%,and the total combined amounts in the range of 84.76～132.26 ng and 91.68～133.83 ng,respectively,demonstrating good accuracy and precision.This study demonstrated a beneficial protocal to solve the problem of real-time monitoring of IAC column capacity in the IAC-HPLC,which also provided a new idea for improving the accuracy and precision of the IAC-HPLC analysis method in the field of food safety.