Development And Validation Of A Novel Online Monitoring Method For The Capacity Of Immunoaffinity Columns Of Ochratoxin A Mimic Peptides And Analogues

Ochratoxins A（OTA）,a mycotoxin mainly produced by Aspergillus and Penicillium,is a nephrotoxic,hepatotoxic,neurotoxic,carcinogenic and teratogenic substance that poses a major threat to human health and food safety.The detection of OTA in food is an important tool to stop OTA-contaminated food from entering the market.In the food safety national standard"GB 5009.96-2016 Determination of Ochratoxin A in Food",Immunoaffinity column-High-performance liquid chromatography（IAC-HPLC）is listed as the first method for the detection of OTA due to its high sensitivity and accuracy.Since the column capacity of the immunoaffinity column（IAC）has a great influence on the accuracy of the assay,it needs to be monitored.However,the current sampling monitoring method of column capacity has certain shortcomings,so it is important to study a method that can monitor the IAC column capacity in real time to improve the accuracy of the assay results.In this study,the mimetic peptide of OTA and the structural modifier of OTA（OTA-R）were obtained by phage random peptide library technique and chemical modification method of OTA molecule,to explore the method of using them as tracers to establish real-time monitoring of immunoaffinity column capacity,to study the law of its application in immunoaffinity column coupled with high performance liquid chromatography,and to further investigate the IAC-HPLC internal standard analysis method.The main studies are as follows.1.Screening and validation of OTA mimetic peptideUsing the phage random line 7 peptide library as the ligand library and the specific monoclonal antibody 2606 of OTA as the target molecule,40 phage clones were screened and validated after three rounds of screening and progressively strict experimental conditions.Among them,36 could bind specifically to the anti-OTA2606 monoclonal antibody and were verified to be inhibited by OTA by enzyme-linked immunosorbent assay（ELISA）.DNA of thoes clones were extracted and sequenced,yielding 18 different amino acid sequences.After chemical synthesized mimetic peptides were tested for validation in a competitive ELISA,the affinity of the mimetic peptide was too low to establish a real-time monitoring method for column capacity of the immunoaffinity column.2.Synthesis,purification and identification of OTA structural modifiersUsing dicyclohexylcarbodiimide（DCC）as a condensation agent,OTA was reacted with N-hydroxysuccinimide（NHS）in anhydrous tetrahydrofuran to form an active ester,and then purified by reaction with glycine to obtain the OTA structural modifier"（（R）-5-chloro-8-hydroxy-3-methyl-1-oxoisochromane-7-carbonyl）phenylalanylglyci ne"（abbreviated as OTA-R）.The OTA-R was identified by fluorescence spectroscopy,UVspectroscopy and mass spectrometry.A competitive ELISA was established to further test OTA-R.In the competitive ELISA curve of OTA-R,y=-45.58x+42.32,the IC₅₀was 0.679 ng/m L,the concentration range was 0.25-10 ng/m L,and the cross-reactivity rate of OTA-R to OTA standard was 32%.After OTA-R and OTA were sampled simultaneously,the two could be separated and quantified under the national standard OTA HPLC assay conditions.3.Preliminary study of OTA-R as internal standard for establishing HPLC internal standard assayThe OTA-R synthesized in this study has similar structure and properties with OTA,and can be distinguished and quantified by HPLC.The standard curve y=4.661x-0.2396,R²=0.9999,and the concentration range of OTA was 1-50ng/m L.After the spiked recovery experiments in a variety of actual samples of rice,corn and millet,the recoveries were 89.1%-119.3%,and the coefficients of variation were1.04%-10.26%.The internal standard method can effectively reduce the matrix interference and improve the accuracy and precision of the assay.4.Application of OTA structural modifier OTA-R in real-time monitoring of IAC column capacityThe spiked recovery experiments were conducted using OTA immunoaffinity column with a column capacity of 450ng.Different amounts of OTA-R were added to IAC at 200ng,300ng and 450ng,and OTA were subsequently added at 5ng,20ng,100ng and 300ng,respectively.when OTA-R was added at 200ng,the average recovery of OTA ranged from 95.3%to 99.7%,the average recovery of OTA-R ranged from 73.4%to 99.7%,and the total binding amount ranged from 208.1ng to465.3ng;when OTA-R was added at 300ng,the average recovery of OTA ranged from88.6%to 100.5%,and the average recovery of OTA-R ranged from 76.8%-101.8%,and the total binding amount was in the range of 337.6ng-501.2ng;when OTA-R was added at 450ng,the average recovery of OTA ranged from 88.8%-115.4%,the average recovery of OTA-R ranged from 43.9%-90.9%,and the total binding amount was in the range of 419.1ng-480.4ng.Based on the experimental results,it can be concluded that the recovery of OTA can always be maintained above 80%when different doses of OTA-R are added,indicating that the addition of OTA-R does not affect the binding of OTA and IAC;and when the addition of OTA-R is moderate,the column capacity can be effectively monitored in real time by the total binding amount of both.This study solves the problem of real-time monitoring of IAC column capacity in the IAC-HPLC assay of OTA,which not only improved the accuracy and precision of the IAC-HPLC analytical method,but also provided a new idea for the application of mycotoxin structural modifiers in real-time monitoring of column capacity in immunoaffinity columns,as well as provided a high scientific and application value in the field of food safety.