The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits.
For the inhibition assay, VLPs were mixed with saliva samples diluted 1 100 for 90 min at room temperature, placed on shellfish tissues, and incubated overnight at 4°C.
For controls, VLPs were applied to shellfish tissues in phosphate-buffered saline/1% bovine serum albumin instead of a saliva sample diluted in the same buffer.
For the binding assay, lectins were applied to shellfish tissues for 30 min at different concentrations (50, 20, 10, 5, and 1 μ>g/mL).
Antibodies that recognize all types of A determinants (3-3A) or those that are restricted to A type 3/4 determinants bound strongly to shellfish tissues (Table 2).
When these lectins were used in an inhibition assay, only HPA had an inhibitory effect on binding of recombinant VLPs to shellfish tissues.
Antibodies directed against H type 2 (19-OLE,), Ley (12-4LE), or A type 2 epitopes (III-2A-5) showed little staining of shellfish tissues and when positive, staining did not correspond to cells to which VLPs bind.
Beach closures due to unsafe levels of domoic acid in shellfish tissues from the harmful algal bloom Pseudo-nitzschia species are relatively recent phenomena in Puget Sound (WA), with the first closure documented in 2003 [ 56].
UEA-1 bound to shellfish tissues, but only at high concentration (50 μg/mL), whereas 2 other lectins, DBA and HPA, bound at a lower concentration (1 μg/mL) (Table 3).
