Development of a high-affinity anti-domoic acid sheep scFv and its use in detection of the toxin in shellfish.
The potential of immunoassays as high-throughput screening tools for the detection of harmful substances in foods will only be realized when convenient methods are available for production of the high affinity antibodies needed for sensitive assay development. Recombinant antibodies offer advantages over traditional monoclonal antibodies in terms of ease of production, much greater antibody repertoire for selection, and versatility. We describe here the development of recombinant antibodies against the common shellfish toxin, domoic acid (DA), utilizing the sheep immunoglobulin system as an effective method for generating high affinity anti-hapten recombinant antibody fragments. A single-chain antibody fragment (scFv) library was generated from a sheep immunized with DA-bovine serum albumin conjugate, and anti-DA scFvs were isolated by phage-display. Three selected scFvs gave I50s of 2.6 to 58 ng/mL (8.3-186 nM) in competitive enzyme-linked immunosorbent assay (ELISA). Assay optimization with one of these scFvs gave a very reproducible standard curve 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 of crude methanolic extracts of scallop tissues, results obtained correlated well with standard HPLC assay results (R2, 0.90, n = 40; R2, 0.81, n = 34), although ELISA results were lower than HPLC results. Adjusting the cutoff point for DA concentration accordingly from the regulatory 20 mg/kg, the potential of the sheep scFv-based ELISA for use as a screening assay for DA in shellfish extracts was demonstrated.
Shaw
Iain
I
National Diagnostics Centre, National University of Ireland Galway, Galway, Ireland. iain.shaw@nuigalway.ie.
O'Reilly
Aoife
A
Charleton
Margaret
M
Kane
Marian
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D008387:Marine Toxins / Q000032:analysis*; D010047:Ovalbumin / Q000737:chemistry; D019151:Peptide Library; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012756:Sheep; D012758:Shellfish / Q000032:analysis*
D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D008387:Marine Toxins; D019151:Peptide Library; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D012313:RNA; D010047:Ovalbumin; C012301:domoic acid; D007608:Kainic Acid