Development and Preliminary Application of Immunochromatography Test Strips for the Detection of Double Residues of Aflatoxin B1 and Zearalenone

Abstract

Based on scientific demonstration and experimental research, this thesis is devoted to studying development and preliminary application of Immunochromatography Test Strips for the Detection of Double Residues of Aflatoxin B1 and Zearalenone, to the establishment of colloidal gold immunochromatographic test strip for the determination of aflatoxin B1 (AFB1) and zearalenone (ZEN) dual residue, so as to provide technical support for the rapid detection of AFB1 and ZEN dual residue in cereal food and feed and ensure the safety of cereal food and feed. Mycotoxins widely exist in the natural environment, mainly pollute all kinds of grain food and feed, seriously threaten the safety of world food and feed, and do harm to human health and animal husbandry development, which has aroused great concern all over the world. Among the more than 400 mycotoxins reported so far, AFB1 and ZEN are the two most important mycotoxins with wide pollution and great harm. Aflatoxins (AFs) are a toxic secondary metabolite produced by Aspergillus, and produced under natural conditions mainly include AFB1, AFB2, AFG1 and AFG2. Nowadays, the established immunoassay methods include gold immunochromatographic assay (GICA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA) and immunosensor (IS), etc. In contrast, GICA technology has the advantages of fast, simple, on-site operation, multiple detection and large amount of screening samples. It has become an important research topic of AFB1 and ZEN dual residue detection, and shows a broad development prospect, which is also the purpose of this study. The preparation of monoclonal antibodies (mAbs) against AFB1 and ZEN with high sensitivity and high specificity is the key to solve the problem of poor quality and unstable source of antibodies. Because the molecular structures of AFB1 and its homologues AFB2, AFG1 and AFG2 all contain dihydrofuran and oxanaphthone, there are only slight differences in the structures of hexacyclic C7-position lactone and pentacyclic C2-position lactone, and the other structures are exactly the same, it is difficult to prepare a high specific antibody for single recognition of AFB1. Similarly, since ZEN and its homologues have slight differences in molecular structure only on the carbonyl or hydroxyl group at C6’-position and the single bond or double bond at C1'-C2'-position of macrolide ring, it is also very difficult to prepare a single highly specific antibody that recognizes ZEN. The synthesis and screening of artificial immunogens are the basis for the preparation of highly specific antibodies, but the specificity of antibodies not only depends on the physical and chemical properties of the immunogen, but is also closely related to immunization methods and antibody screening methods. This study tries to use small dose (30 μg/mL), long immunization interval (4 weeks), multiple sites on the back (4 to 6 points), and multiple frequency (5 times) immunization methods. At the same time, try to use heterologous indirect non-competitive ELISA (inELISA) and heterologous indirect competition ELISA (icELISA) screen mAbs, and prepare the desired AFB1 mAbs and ZEN mAbs by the above methods. The establishment of colloidal gold immunochromatographic test strip detection method is the path to solve the rapidity, simplicity, on-site detection and multivariate detection of detection technology. AFB1 mAbs and ZEN mAbs with high-sensitivity and high-specificity are used as the labeling target, colloidal gold is used as the labeling material, two mAbs are labeled with nano-gold and two detection lines are set to realize the dual residue detection of AFB1 and ZEN. By comparing and analyzing the sensitivity of gold labeled antigen competition mode and gold labeled antibody competition mode, the gold labeled antibody competition mode is selected in this paper. In this study, colloidal gold is prepared by trisodium citrate reduction method, gold labeled antibodies are prepared by mey's series stabilization method, and the detection conditions of test strip are optimized, including nitrocellulose membrane selection, gold labeled pad selection, sample pad selection, optimal concentration combination of gold labeled antibody and coated antigen, determination of methanol concentration in sample treatment solution, etc. Finally, the AFB1 and ZEN dual residue colloidal gold immunochromatographic test strip detection method was established, its performance is determined and preliminarily applied, and confirmed by HPLC-MS/MS, so as to provide technical support for the detection of AFB1 and ZEN dual residue in food and feed. Research has established the method for the preparation and identification of artificial immunogens and coating antigens to obtain high-specific antibodies against AFB1 and ZEN. According to the molecular structure characteristics of AFB1, the carbonyl at C1, the active hydrogen at C2, the hydroxyl at C3, and the C3-C4 bifuran ring are selected as the active sites or active groups, and 6 kinds of artificial immunogen AFB1-BSA and coating antigen AFB1-OVA synthesis methods, including oxime active ester method (OAE), methylation of ammonia (MOA), mixed anhydride (MA), semi acetal (SA), epoxide (EP) and enol ether derivation (EED), have been established. According to the molecular structure characteristics of ZEN, the carbonyl at C6', the active hydrogen at C7', the hydroxyl at C2, and the C5 at the benzene ring are selected as the active sites or active groups, and 5 kinds of artificial immunogens ZEN1-BSA and coating antigen ZEN-OVA synthesis methods, including oxime active ester method (OAE), condensation mixed anhydride method (CMA), formaldehyde method (FA), 1,4-butanediol diglycidyl ether method (BDE) and amino glutaraldehyde method (AGA), have been established. The artificial immunogens are identified by UV, SDS-PAGE and animal immune effect. OAE method is the best method to prepare AFB1 high specific antibody, and AGA method is the best method to prepare ZEN high specific antibody. Research has established animal immunization method and screening method for positive hybridoma cell lines. The factors affecting animal immune effect include dosage, injection route, time interval, adjuvant use, immunization times and individual differences, among which, immunization dose and time interval are the key factors. After continuous attempts, we have reached the same conclusion as some scholars that low-dose immunogen can induce the production of specific antibodies, and a longer time interval can improve the affinity of antibodies. Therefore, this study established a low-dose (30 μg/mL), long-term interval (4 weeks), multiple points on the back (4-6 points), and multiple frequency (5 times) animal immunization methods. This study successfully established the screening method for positive hybridoma cells, including 4 steps. The first is to use homologous inELISA to determine the antibody titer to correctly evaluate the immunogenic immunoreactivity; the second is to use heterologous inELISA to determine the antibody titer to evaluate the recognition of antibodies; the third is to use a heterologous icELISA to determine the IC50 value of the antibody to AFB1 to correctly evaluate the sensitivity of the antibody; the fourth is to use a cross-reactivity test to determine the cross-reactivity between the antibody and hapten analogs to correctly evaluate the antibody specificity. Using the above method, 3 positive hybridoma strains of AFB1 mAb 2A11, 2F6 and 3G2, and 2 positive hybridoma strains of ZEN mAb 2B6 and 4D9 are screened. The mAbs are prepared by inducing ascites in vivo, and the immunological characteristics of each mAb are identified, including karyotype identification by colchicine blocking method, the class and subclass determination by mouse mAb homotype kit, the stability determination by hybridoma cell passage culture method, the affinity constant (Ka) determination by batty saturation method, the sensitivity determination by icELISA, and the specificity determination by cross-reaction test. Research has established AFB1 single residue (ZEN single-residue) and AFB1 and ZEN dual residue colloidal gold immunochromatographic test strip detection methods and performance identification methods. The AFB1 single residue colloidal gold immunochromatographic test paper is constructed with nano-gold labeled AFB1 mAb, and the test strip parameters are optimized. Similarly, ZEN single residue colloidal gold immunochromatographic test paper is assembled. AFB1 mAb and ZEN mAb are labeled with nano-gold, and a certain proportion of mixed gold labeled mAbs are configured according to the detection sensitivity of single residual colloidal gold immunochromatographic test strip; The corresponding immunogens AFB1-BSA (OAE) and ZEN-BSA (AGA) are used to set up two detection lines on the nitrocellulose (NC) membrane, and the dual residue colloidal gold immunochromatographic test strip of AFB1 and ZEN is assembled and established. The performances of the prepared colloidal gold immunochromatographic test strip are determined, including the visual detection limit by visual method, the machine-readable detection limit and IC50 value by the test strip reader scanning method (BioDot-TSR3000 strip reader), the stability determination by intra batch and inter batch tests, and the validity period determination according to the test results. The 6 immunogens of AFB1 were identified by UV and SDS-PAGE, and the results showed that the 6 immunogens were synthesized successfully, and the molecular binding ratios of AFB1 to BSA were 8.64∶1 (OAE), 6.88∶1 (MOA), 10.78∶1 (MA), 4.46∶1 (SA), 6.38∶1 (EP) and 2.31∶1 (EED), respectively. Balb/c mice were immunized with 6 immunogens of AFB1. The results showed that AFB1-BSA (OAE) had the best effect among the 6 immunogens. The titer of AFB1 pAb reached 1:(1.6×103), and the IC50 value was 10.14 μg/kg. AFB1 pAb had the strongest specificity, could recognize AFB1 100%, and its CR values with AFB2, AFG1 and AFG2 were 6.32%, 3.76% and less than 1.0%, respectively. Similarly, the 5 immunogens of ZEN were identified by UV and SDS-PAGE, and the results showed that the 5 immunogens were synthesized successfully, and the molecular binding ratios of ZEN and BSA were 17.2:1 (OAE), 14.6:1 (CMA), 9.7:1 (FA), 8.3:1 (BDE) and 11.6:1 (AGA), respectively. Among the 5 immunogens of ZEN, ZEN-BSA (AGA) had the best immune effect. The titers of ZEN pAb reached 1:(1.6×103), and the IC50 value was 18.77 μg/kg. It had the strongest specificity and could recognize ZEN 100%, and its CR values with α-ZAL, α-ZAL, α-ZOL, β-ZOL, ZON were 1.48%, 1.36%, 3.57%, 1.65% and 4.86%, respectively. Therefore, this study screened out that the OAE method was the best method for preparing AFB1 highly specific antibodies, and the AGA method was the best method for preparing ZEN highly specific antibodies. The immunological characteristics of the selected AFB1 mAb 2A11, 2F6 and 3G2 hybridoma cell lines were identified. The results showed that the best was 2A11, which belonged to the IgG1 subtype with kappa light chain, and could stably secrete antibodies after 5 passages. The titers of AFB1 mAb in cell culture supernatant was 1: (6.4×102) and in ascites was 1: (5.12×105) by inELISA. Its Ka was 1.05×109 L/moL, and its IC50 value for AFB1 was 6.28 μg/kg. It could recognize AFB1 100%, and its CR values with AFB2, AFG1 and AFG2 were 4.35%, 2.30% and less than 1.0%, respectively. Similarly, two positive hybridoma cell lines of ZEN mAb 2B6 and 4D9 were identified, and the results showed that the best of them was 2B6, which belonged to the IgG1 subtype with kappa light chain, and could stably secrete antibodies after 5 passages. The titers of ZEN mAb in cell culture supernatant was 1: (5.12×102) and in ascites was 1: (5.12×105) by inELISA. Its Ka was 7.69×109 L/moL, and its IC50 value for ZEN was 10.38 μg/kg. It could recognize ZEN 100%, and its CR values with α-ZAL, α-ZAL, α-ZOL, β-ZOL, and ZON were 1.52%, 1.28%, 2.64%, 1.83%, and 4.27%, respectively. Therefore, this study produced high-titer, high-sensitivity and high-specificity AFB1 mAb and ZEN mAb, which could be used for immunoassays for the detection of AFB1 and ZEN residues. The performances of AFB1 (or ZEN) single residue test strip and AFB1 and ZEN dual residue test strip were measured. The results showed that the visual LOD of AFB1 single residue test strip was 1.0 µg/L (5.0 µg/L for ZEN), the machine-readable LOD was 0.24 µg/L (1.51 µg/L for ZEN), and the machine-readable IC50 value was 1.11 µg/L (4.97 µg/L for ZEN). There was no CR with other compounds. The results of 6 batches of AFB1(or ZEN) single residue strip were measured and showed good stability. The validity period was 6 months at 4 ℃ in refrigerators and 25 ℃ room temperature. The visual LODs of AFB1 and ZEN dual residue test strip were 1.0 ng/mL and 5.0 ng/mL for AFB1 and 5.0 ng/mL respectively, and the machine-readable LODs for AFB1 and ZEN were 0.23 µg/L and 1.53 µg/L respectively, and the machine-readable IC50 value for AFB1 and ZEN were 1.15 µg/L and 4.91 µg/L respectively. The test results of 6 batches of AFB1 and ZEN dual residue test strip of different batches were completely consistent and showed good stability. The validity period was the same as the single residue test strip. The AFB1 and ZEN dual residue test strip were used for preliminary application and verified by HPLC-MS/MS. The 20 known positive samples including 12 AFB1 positive samples and 8 ZEN positive samples were tested. The results showed that the positive coincidence rate was 100%. A total of 60 natural samples of corn, rice, flour, and feed were tested. 39 positive samples were detected, including 22 positive samples for AFB1 and 17 positive samples for ZEN, which were consistent with the HPLC-MS/MS detection results, with a coincidence rate of 100 %. Thus, the results of this study focused on the synthesis of ideal immunogens, which solved the problem of poor immunogenicity of AFB1 and ZEN; preparation high-titer, high-sensitivity and high-specificity AFB1 mAbs and ZEN mAbs, which solved problems of the unstable source of antibodies and unstable quality of antibodies; the establishment of AFB1 (or ZEN) single residue and AFB1 and ZEN dual residue detection test strip, which solved the problems of rapidity, simplicity, multiple detection and on-site detection; preliminary application and verification by HPLC-MS/MS of AFB1 (or ZEN) single residue and AFB1 and ZEN dual residue detection test strip, which solved the problems of practicability and reliability. Finally, technical support was provided to realize the rapid detection of AFB1 and ZEN dual residue and ensure the safety of food and feed. We recommend using the materials of the dissertation work when studying the courses " Veterinary microbiology", Veterinary sanitary examination" for masters of the Faculty of Veterinary Medicine of Sumy NAU. And for the courses "Veterinary microbiology “for masters of the Henan Institute of Science and Technology (HIST).