H . PREPARATIONS BEFORE THE ANALYSIS a) Preparation of buffers and reagents 1. Washing buffer (PBS-T; 0.05% Tween 20 in PBS): Dissolve one tablet (C) in distilled water and dilute to 500 mL. May be stored at 4ºC for one week. 2. Extraction solution (50% methanol in water): Prepare sufficient solution for the required number of samples by mixing equal volumes of methanol and distilled water. Prepare fresh each day. 3. Standard/Sample buffer (10% methanol in PBS-T): Mix 5 mL of methanol with 45 mL of Washing buffer. May be stored for 2-3 days at room temperature. 4. Antibody-HRP buffer (1% ovalbumin in PBS-T): Add 6 mL of Washing buffer to 60 mg of ovalbumin (vial F). Prepare fresh for each assay. b) Preparation of Domoic acid calibration solutions The 10-point calibration curve is freshly prepared using standard dilutions in the range of 10 000 – 0.16 pg DA/mL: 1. Prepare one Eppendorf tube containing 450 μL Standard/Sample buffer (10% methanol in PBS-T) - “tube 1”, and 9 Eppendorf tubes containing 300 μL Standard/Sample buffer - “tubes 2-10”. 2. Add 50 μL of the DA standard (100 ng/mL, vial D) to tube 1 and vortex, to obtain a 10 ng/mL DA solution. 3. Transfer 125 μL of the 10 ng/mL solution (tube 1) to tube 2 and vortex. 4. Complete the 3.4-fold dilution series by transferring 125 μL from tube 2 to tube 3 and vortex. Repeat this step for all tubes 3-10 (see Fig. 3).  

F I G U R E 3 .DOMOIC  ACID STANDARD DILUTION SEQUENCE

   
I . PREPARATION  OF  SHELLFISH  SAMPLES   a) Extraction of DA from shellfish samples Shellfish flesh should be prepared as a finely blended homogenate. Preferably analyse fresh, but it may be stored frozen at -20°C for up to 14 days before use. 1. Prepare shellfish homogenate in a high speed blender, from no less than 50 g shellfish flesh. 2. Accurately weigh 4 g into a 50 mL centrifuge tube. 3. Add 16 mL of Extraction solution (50% methanol). 4. Mix well by vigorous shaking on vortex for 1 min. 5. Centrifuge at 3000xg for 10 minutes at room temperature. 6. Retain the supernatant for further dilution prior to analysis. The extracts can be stored at -20˚C for up to 14 days, although with a possible reduction in DA content.   b) Dilution of shellfish sample extracts 7. Prepare dilutions of the shellfish extract in Standard/Sample buffer (10% methanol in PBS-T) as follows:               1:20 dilution:       50 μL shellfish extract           + 950 μL buffer         1:200 dilution:      50 μL of the 1:20 dilution        + 450 μL buffer         1:2000 dilution:     50 μL of the 1:200 dilution       + 450 μL buffer         1:20 000 dilution:   50 μL of the 1:2000 dilution      + 450 μL buffer Cap and vortex each dilution before proceeding to the next dilution step. 8. Analyze the sample dilutions according to the DA concentration range of interest (see Table 1), to give absorbance values within the calibration curve working range. It is recommended to analyze shellfish extracts diluted at 1:20 000 dilutions to comply with EC directive 2002/226/EC, for the quantification of DA up to the maximum permitted level at 20 mg/kg.   T A B L E 1 : SHELLFISH EXTRACT DILUTION FOR QUANTIFICATION OF DA