| Abstract
| - The potential of immunoassays as high-throughput screening tools for the detection of harmful substances in foodswill only be realized when convenient methods are available for production of the high affinity antibodies neededfor sensitive assay development. Recombinant antibodiesoffer advantages over traditional monoclonal antibodiesin terms of ease of production, much greater antibodyrepertoire for selection, and versatility. We describe herethe development of recombinant antibodies against thecommon shellfish toxin, domoic acid (DA), utilizing thesheep immunoglobulin system as an effective method forgenerating high affinity anti-hapten recombinant antibodyfragments. A single-chain antibody fragment (scFv) librarywas generated from a sheep immunized with DA-bovineserum albumin conjugate, and anti-DA scFvs were isolated by phage-display. Three selected scFvs gave I50sof 2.6 to 58 ng/mL (8.3−186 nM) in competitive enzyme-linked immunosorbent assay (ELISA). Assay optimizationwith one of these scFvs gave a very reproducible standardcurve with a range of 0.3 to 5.6 ng/mL (1.0 to 17.9 nM),a mean limit of quantification (LOQ, defined as the I20)of 0.5 ng/mL (1.6 nM), and a mean I50 of 1.2 ng/mL(3.9 nM). When the assay was used for the analysis ofcrude methanolic extracts of scallop tissues, resultsobtained correlated well with standard HPLC assay results(R2, 0.90, n = 40; R2, 0.81, n = 34), although ELISAresults were lower than HPLC results. Adjusting the cutoffpoint for DA concentration accordingly from the regulatory20 mg/kg, the potential of the sheep scFv-based ELISAfor use as a screening assay for DA in shellfish extractswas demonstrated.
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