Development of a High-Affinity Anti-Domoic Acid Sheep scFv and its Use in Detection of the Toxin in Shellfish

Abstract

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.

Biosensors for disease biomarker detection

“…This reduces the production cost while maintaining its functionality (Peterson et al , 2006;Whittington et al , 1998). The use of recombinantly produced single chain variable fragment (scFv) antibodies as immunoassay capture and display biomolecules has generated considerable interest (Dominguez et al , 1999;Giuliani et al , 1989;Jaaskelainen et al , 2008;Luo et al , 1998;Shaw et al , 2008;Shen et al , 2008;Wang et al , 2008). As shown in Fig.…”

Chemiluminescence immunoassay

“…Shaw et al [21] described the development of rAbs (scFv) against the common shellfish toxin, domoic acid (DA), utilizing the sheep immunoglobulin system as an effective method for generating high-affinity anti-hapten rAb fragments.…”

Integrating scFv into xMAP Assays for the Detection of Marine Toxins

“…A viable alternative is recombinant antibodies including single-chain variable fragment (scFv), and single domain antibodies (sdAb) [36]. Recombinant constructs (scFv) that consist of linked heavy and light variable domains that make up the binding domains of conventional antibodies have been produced for the sensitive detection of DA, as well as a number of other biotoxins [37,38]. Recombinantly produced binding elements serve to “preserve” mAbs as they can be easily recreated from their sequence information, thereby reducing variability and eliminating issues with loss of cell lines.…”

“…This work utilized the previously described anti-DA binding fragment [37] in conjunction with an anti-STX binding domain that was synthesized from the sequence of an anti-STX mAb [15]. In addition to detection in buffer, we show the utility of the assay in shellfish matrices.…”