The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA

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 × 10 ⁸ 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 IC ₅₀ 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.