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Improved detection of domoic acid using covalently immobilised antibody fragments.

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.

SUBMITTER: Hortiguela MJ

PROVIDER: S-EPMC3705377 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Improved detection of domoic acid using covalently immobilised antibody fragments.

Hortigüela María J MJ Wall J Gerard JG

Marine drugs 20130314 3

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 an ...[more]

PMID: 23493076

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Project description:Domoic acid (DA) is a naturally produced neurotoxin synthesized by the marine diatom genus Pseudo-nitzschia. DA accumulates in filter-feeders such as shellfish, and can produce severe neurotoxicity when contaminated seafood is ingested. DA poisoning is a significant public health concern, and though seafood regulations have effectively minimized the human risk of severe acute poisoning, the effects of exposure at asymptomatic levels are poorly understood. The objective of this study was to determine the effects of exposure to symptomatic and asymptomatic doses of DA on gene expression patterns in the zebrafish brain. We exposed adult zebrafish to either a symptomatic (1.1 ± 0.2 μg /g) or an asymptomatic (0.31 ± 0.03 µg DA/g fish) dose of DA by intracelomic injection and sampled at 24, 48 and 168 h post-injection. Transcriptional profiling was done using Agilent and Affymetrix microarrays. Our analysis revealed distinct, non-overlapping changes in gene expression between the two doses. We found that the majority of transcriptional changes were observed at 24 hours post-injection with both doses. In response to symptomatic dose exposure, we observed 328, 5, and 1 genes were differentially expressed at 24, 48 and 168 hours post-injection, respectively. In contrast, 136, 12, and 28 genes were differentially expressed in the asymptomatic dose at the same time points. Pathway analysis revealed symptomatic DA exposure affected genes associated with pathways including cell adhesion, inflammation, and amyloid proteins. Among the pathways enriched among genes differentially expressed with asymptomatic exposure were circadian rhythms and endocrine signaling. Overall, these results suggest that transcriptional responses are specific to the DA dose and that asymptomatic exposure can cause long-term changes. Many of the differentially expressed genes are important players in neuronal, neuroimmune and neuroendocrine function. Further studies are needed to characterize the potential downstream neurobehavioral impacts of DA exposure.

Dataset Information

Improved detection of domoic acid using covalently immobilised antibody fragments.

Publications

Improved detection of domoic acid using covalently immobilised antibody fragments.

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