Development of monoclonal antibodies to domoic acid for the detection of domoic acid in blue mussel (mytilus edulis) tissue by ELISA

Monoclonal antibodies were developed against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. Three murine monoclonal antibodies, 4D1D, 9B10D. and 7D6C, were obtained from hybridoma clones. They were derived by fusion of mouse myeloma cells with mouse immune spleen cells derived from animals immunised with domoic acid-KLH conjugate. The hybridoma cell line 4D1D was adjusted to serum free medium. It produced monoclonal antibodies of high selectivity and sensitivity. Mass production was carried out with two different fermenter systems. Monoclonal antibody levels up to 0.7 mg/ml were obtained. No cross-reactivities against similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid could be detected. A detection limit of 4 mug/I DA with a test midpoint (IC50) of 10 mug/1 DA was achieved with a competitive enzyme immunoassay applying a streptavidinbiotin amplification system (ABC system). Moreover this test format was well suited to detect DA in spiked mussel extracts. To protect consumers from intoxication from DA, most countries have set a regulatory level of 20 mug/g mussel tissue. This regulatory level allows a maximum 5000-fold dilution of the samples containing this DA level.