Abstract
Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006–0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56 ± 2.8% with the coefficient variation of 0.01–0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.
Highlights
Marine toxins are noxious, poisonous and hazardous substances, which can be found as short peptides, proteins or secondary metabolites
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to assay successful Domoic acid (DA)-protein conjugation, and the result showed that DA-bovine serum albumin (BSA) and DA-OVA conjugates moved faster than carrier proteins alone, demonstrating that DA was successfully conjugated (Figure 1A,B)
Ultraviolet spectral scanning curves were further obtained, and the shifts in wavelength were showed by DA-BSA
Summary
Poisonous and hazardous substances, which can be found as short peptides, proteins or secondary metabolites. The toxins are produced by a range of phytoplankton taxa and other related microorganisms causing deadly diseases and lethal impacts on the health of marine mammals, birds and humans [1]. DA is a potent toxin, marine biotoxin, and hazardous to the human health. It is produced by the diatomic algal genus of Pseudo-nitzschia and other related species. It is transmitted to the marine mammals and to the humans via food chain by the consumption of contaminated seafood products primarily including species related to the shellfish [3,5]
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