Abstract
Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.
Highlights
Of the 5000 phytoplankton species known to date, approximately 300 can give rise to algal blooms and 40 species, which produce marine toxins, harmful algal blooms (HABs)
The single-chain Fv (scFv)-encoding genes, containing an N-terminal ompA leader peptide for secretion of the translated polypeptide to the Escherichia coli periplasm, an adjacent hexahistidine tag for detection and purification of the scFv and the relevant cysteine-containing tag at the 3′-end, were generated and cloned into the pIG6 vector to express the proteins shown in Figure 1B, followed by confirmation of construct sequences prior to carrying out protein expression
The VL C-terminal lysine residue 276 to which the cysteine-containing linkers were attached is labelled. (B) Schematic of the scFvs expressed in the study. scFv: single-chain Fv fragment containing the 2H12 VH and VL domains; scFv-cys I: scFv containing additional cysteine residue in a 6-amino acid C-terminal peptide linker; scFv-cys
Summary
Of the 5000 phytoplankton species known to date, approximately 300 can give rise to algal blooms and 40 species, which produce marine toxins, harmful algal blooms (HABs). Domoic acid (DA) is a water-soluble amino acid and the principal cause of amnesic shellfish poisoning (ASP) in humans It is produced by diatoms of the genus Pseudo-nitzschia and accumulates mainly in the digestive glands of filter-feeding shellfish and fin fish such as anchovies and sardines that feed on the phytoplankton that produce the toxin (reviewed in [4]). The main platforms used to detect DA in shellfish samples are bioassays and biochemical or chemical approaches [11,12] In the former group, the commonly used mouse toxicity assay raises obvious ethical concerns and is expensive, not sufficiently sensitive to meet regulatory needs [11] and subject to false positives and negatives [13]. The strategy has broad potential application in biosensing of marine toxins
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