Monoclonal antibody-based enzyme immunoassay for domoic acid by using hapten-protein conjugates obtained at the nanomolar level in a reversed micellar medium.

Publication Type  Book Chapter
Year of Publication  2001
Authors  Branaa, P.; Naar, J.; Chinain, M.; Pauillac, S.
Book Title  Harmful Algal Blooms
Publisher  Hallegraeff G.M., Blackburn S.I., Bolch C.J. and Lewis R.J. , Eds - Intergovernmental Oceanographic Commission of UNESCO
Pages  272-5
Language  eng
Abstract  

A competitive enzyme-linked immunosorbent assay (ELISA) to measure domoic acid (DA) has been developed. DA-protein conjugates were prepared via the mixed anhydride method of Erlanger performed in a reversed micellar medium using 0.32-0.64 µmol of DA in a 100-fold molar excess relative to protein. Two specific monoclonal antibodies (MAbs), 1D12 and 3E1 were produced by hybridoma technology following immunization of a BALB/c mouse with a DA-bovine serum albumin conjugate. No significant cross-reactivity was observed with either glutamic, aspartic or kainic acids or proline. MAb 1D12 enabled the accurate and reproducible detection of DA levels in spiked mussel extracts pre-cleaned through a solid phase extraction column. A very good correlation (r2 = 0.96 ; n=6) was observed between the actual amounts of DA added and amounts detected. The working range achieved with this ELISA (0.3 - 3µ/g of original mussel tissue) strongly suggests its potential as alternative assay for routine monitoring of shellfish (Maximum Permitted Level = 20µ/g of shellfish tissue).

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