Application Of Zearalenone Mimetic Peptides And Structural Analogues In Immunoaffinity Columns

Zearalenone(ZEN)is one of the most widely distributed mycotoxins in the world,which has estrogen-like effects and seriously affects animal and human health,so the accurate detection of ZEN is crucial.Immune affinity column High performance liquid chromatography(IAC-HPLC),the first method of national standard,can accurately characterize and quantify ZEN.The column capacity of IAC affects the accuracy of the detection results.The current determination of IAC column capacity is done by sampling,and the capacity of each affinity column cannot be determined,which poses a hidden danger to the assay results.Therefore,in order to explore the method of realtime monitoring of IAC column capacity,this study investigated the tracers for realtime column capacity monitoring by two technical routes: biological screening and chemical modification.Then the acquired mimetic peptides obtained by biological screening and the chemically synthesized ZEN analogues are examined for the realtime monitoring of ZEN immunoaffinity column capacity.The relevant studies are summarized as follows.1.Screening and validation of ZEN mimetic peptidesThrough three rounds of panning of phage ring 7 and line 7 random peptide libraries,three phage clones with different DNA sequences were panned in the screening results of line 7 peptide library,and the competition curves of the three sequences were established respectively,and it was found that their linear ranges were between 10-1000 pg/m L,with basically similar competition inhibition curves,among which the 50% inhibition binding concentration(IC50)of clone L1 Among them,the lowest IC50 was 74 pg/m L for clone L1;In the screening result of peptide library of ring 7,11 phage clones with different DNA sequences were selected,among which 9phage clones had a common sequence: tryptophan-tyrosine-arginine-leucine.Z7 had the lowest IC50 of 215рg/m L.2.Synthesis and validation of ZEN mimetic peptideThe insertion amino acid sequences of clones L1 and Z7 were chemically synthesized to obtain the mimetic peptide,and the mimetic peptide was applied in ELISA competition experiments to optimize the conditions: the mimetic peptide amidated at the C-terminus with the best linearity and the lowest IC50 under the conditions of lysis solution of PBS,the competitive binding time was 10 min,and the linear range of the C-terminus amidated mimetic peptide L1 was 8-350 μg/m L,IC50 was 201 μg/m L;The linear range of the C-terminal amidated mimetic peptide Z7 was3-100 μg/m L,and the IC50 was 20 μg/m L,which had low affinity with ZEN antibody,so it could not be used to establish a real-time column capacity monitoring method for immunoaffinity columns.3.Synthesis,purification and identification of ZEN structural analoguesTwo structural analogues of ZEN(α-ZEL-G and β-ZEL-G)were synthesized by esterification of two derivatives of ZEN,α-zearalenol(α-ZEL)and β-zearalenol(β-ZEL),with glutaric anhydride.The samples were purified and identified by fluorescence,UV and high-resolution mass spectrometry,and the structural properties were in accordance with the expectation;The competitive ELISA curves of α-ZEL-G and β-ZEL-G were established with IC50 1.3 ng/m L and 10 ng/m L,respectively.The cross-reactivity of α-ZEL-G and β-ZEL-G with ZEN standard was 153.85% and20.00%,indicating that the affinity of α-ZEL-G with the antibody was slightly higher than that of ZEN,while the affinity of β-ZEL-G with the antibody was lower than that of ZEN.The recoveries of α-ZEL-G and β-ZEL-G were 93.29% and 91.41%,respectively after being put into the ZEN IAC,which proved that α-ZEL-G and β-ZEL-G could be retained by the immunoaffinity column,which suggested that α-ZELG,β-ZEL-G could be further studied as tracers for ZEN IAC column capacity.4.Application study of ZEN structural analogues in IAC-HPLCThe results of the study of α-ZEL-G and β-ZEL-G as tracers for real-time monitoring of the column capacity of ZEN IAC showed that: even if 6000 ng of β-ZEL-G was sampled simultaneously with 2000 ng of ZEN,the binding of ZEN to IAC was not affected,and the recovery of ZEN was more than 80%;while 6000 ng of α-ZEL-G affected the binding of ZEN to The recovery of 2000 ng of ZEN was less than60%,indicating that β-ZEL-G,which has lower affinity with antibodies,is more suitable for column volume real-time monitoring.The column volume real-time monitoring method established by β-ZEL-G was applied to the sample detection of the three most commonly contaminated cereals(corn,rice and wheat)by ZEN.The results showed that the spiked ZEN recoveries ranged from 92.84% to 129.12% with the relative standard deviations(RSD,n=5)of 0.92%-13.74% for wheat,from 93.82% to115.64% with the relative standard deviations(RSD,n=5)of 1.81%-6.07% for corn,from 92.51% to 108.56% for rice,and the relative standard deviations(RSD,n=5)were3.12%～9.32%,and the real-time monitoring of column capacity was achieved without affecting the accuracy and precision of the assay.