Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

Abstract

Abstract:A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices. Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation AbstractA comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

“…Additionally, removing the major framework regions of the parental mAb and adding a linker peptide, which lead to the binding site structure of scFv somehow different from that of the parental mAb, generally, had minimal effect on the recognition capability of the variable regions in our study. This finding agrees with previous reports [ 27 , 39 , 40 ].…”

“…These results are consistent with previous experiences, where high-quality scFv is highly dependent on the property of the hybridoma cell line and can be constructed via homologous recombination of VH or VL gene fragments [ 22 ]. Thus, the significantly different binding capability between the anti-triazophos scFv-8C10 and a broad-specific scFv [ 27 ] might stem from the different CDRs in variable fragments. Additionally, removing the major framework regions of the parental mAb and adding a linker peptide, which lead to the binding site structure of scFv somehow different from that of the parental mAb, generally, had minimal effect on the recognition capability of the variable regions in our study.…”

“…Apart from scFvs for biomolecules, several functional scFvs against small molecular pollutants have been developed. These include biotoxins such as fumonisin B1 [ 20 ], Cry1B toxin [ 21 ], and aflatoxin [ 22 ]; insecticides, such as parathion [ 23 ], chlorpyrifos-ethyl [ 24 ], fenitrothion [ 25 ], and carbaryl [ 26 ]; and fungicides, such as tetraconazole, imazalil, and thiabendazole [ 27 ]. A broad specificity recombinant antibody was developed for OPPs, which exhibited a cross reaction to triazophos with IC 50 of 25.3 ng/mL [ 28 ].…”

Evaluation of a time efficient immunization strategy for anti-PAH antibody development

“…Monoclonal antibodies (mAbs) have been used during the last decades as immuno-analytical tools for the detection of low molecular weight contaminants in food and the environment (1). The advantages of antibody-based immunoassays are that they are quick, inexpensive, simple to perform, and reasonably portable; they can also be highly sensitive and selective (2).…”

“…The resulting “hybridomas” are evaluated by immunological screening procedures, such as competitive ELISAs for the desired affinities and specificities (8) and are able to produce a uniform population of antibodies with the preferred characteristics. This whole process is time-consuming (1) and it usually requires 3 to 6 months to produce suitable mAbs (9). Typically during the PAH mAb development procedure, half of the time is used to monitor the sera for a satisfactory titer prior to fusion.…”