A competitive enzyme-linked immunoassay for domoic acid determination in human body fluids

A polyclonal antiserum was raised in mice against domoic acid. Two of three immunogens consisted of domoic acid coupled to ovalbumin (OVA) and keyhole limpet haemocyanin at molar ratios of 47:1 and 44:1, respectively using a carbodiimide reaction. Titres of both antisera exceeded 1/35,000 against domoic acid coupled to the non-relevant carrier. Domoic acid was also conjugated to bovine serum albumin at a molar ratio of 30:1 using N-hydroxysuccinimidyl-4-azidobenzoate, a photoreactive compound. This immunogen, however, produced no measurable serum titres against domoic acid. The antiserum produced against the OVA conjugate displayed the highest affinity for free domoic acid in competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, this antiserum preparation did not significantly cross-react with glutamic acid, aspartic acid, the structural analogue kainic acid, or the paralytic shellfish toxin, saxitoxin. The competitive ELISA was used to quantify domoic acid concentrations in human body fluids spiked with pure domoate. The lower limits of accurate domoic acid determinations in competitive ELISA were 0.2 micrograms/ml in urine, 0.25 micrograms/ml in plasma and 10 micrograms/ml in milk. It was concluded that the competitive ELISA described herein could be used to quantitate directly the concentration of domoic acid in the body fluids of individuals with amnesic shellfish poisoning.