德国IBL ASP ELISA试剂盒使用说明（四）
J. PREPARATION OF DA SAMPLES FROM OTHER MATRICES
a) Preparation of algal samples
The analysis of algal samples will depend on the amount of algae (cells/mL) and the
amount of DA present in the algae and in the seawater or culture medium.
1. Algal samples are typically collected by gently vacuum filtering seawater
samples (10 – 100 mL) through a glass-fibre filter (GF/C, 47 mm) until the water
meniscus disappears. Do not suck hard or to dryness. To determine the amount
of DA in the seawater, also collect the flow-through filtrate and analyze this
fraction as described in “Preparation of seawater samples”.
2. Carefully lift the edge of the filter with tweezers, roll into a cylinder and insert
in a 15 mL Falcon tube. Cap and snap-freeze in dry ice as soon as possible after
filtering. The tubes can be stored at -20˚C for up to 2 weeks prior to analysis.
3. Add 10 mL of 20% methanol (1 + 4 v/v methanol plus distilled water) to the
filter in the tube.
4. Cap and vortex for 1 minute. Centrifuge at 3000xg for 10 minutes at room
temperature.
5. Dilute the algal extracts in Standard/Sample buffer before analysis. The
number of cells collected and amount of DA in the samples may vary
considerably. It is recommended that the sample dilutions tested do not
contain more than the equivalent of 100 cells/mL.
b) Preparation of seawater samples
1. Centrifuge the seawater for 10 minutes at 3000xg to remove debris from the
sample solution. Alternatively, filter the seawater through a 0.45 μm filter.
2. Dilute the cleared seawater samples in Standard/Sample buffer. For seawater
samples a minimum dilution of 1:25 is recommended.
K . ASSAY PROCEDURE
a) Incubation of standards and samples with antibody
Equilibrate pre-coated plate strips and all reagents to room temperature before use
(1 hour max). See Figure 4 for a recommended plate layout for either using 4 strips
in 2 rounds of analysis (4A), or all 8 strips at once (4B).
1. Open the packet(s) with pre-coated plate strips gently and place the strips in the
strip frame. Label each strip e.g. A, B, C and D etc.
2. Add 300 μL Washing buffer to each well. Pre-soak the wells for 5-10 minutes.
3. Remove the Washing buffer by inverting the strips over a sink and tap against
a pile of paper towels to remove all the remaining liquid.
4. Add 50 μL Standard/Sample buffer (10% methanol in PBS-T) to each of the
duplicate Amax and Blank wells.
5. Add 50 μL Antibody-HRP buffer (1% ovalbumin) to the Blank wells.
6. Add 50 μL of each DA standard dilution to each of two wells.
7. Add 50 μL of each sample dilution to each of two wells.
8. Shake vial E briefly, and tap the vial gently on a hard surface to ensure that all
the content is in the bottom of the vial. Transfer 0.5 mL (for 4 strip assay) or 1.0
mL (for 8 strip assay) from vial E (concentrated Anti-DA-HRP) to a Falcon type
tube containing 2.5 mL (for 4 strip assay) or 5.0 mL (for 8 strip assay) Antibody-
HRP buffer (1% ovalbumin). Vortex briefly.
9. Add 50 μL of the diluted Anti-DA-HRP conjugate to all wells except the Blank
wells.
10. Seal the strips with the plate sealer (B) and incubate at room temperature (20-
25°C) for 1 hour. Protect from light (e.g. cover with aluminium foil or place in
a drawer).
FIGURE 4A : SUGGESTED PLATE LAYOUT , USING 4 STRIPS, FOR THE QUANTIFICATION OF DA IN 12 SHELLFISH S AMPLES IN 2 SEPARATE ROUNDS .
S1=SAMPLE 1, S2=SAMPLE 2 , ETC.
FIGURE 4A : SUGGESTED PLATE LAYOUT , USING 8 STRIPS, FOR THE QUANTIFICATION OF DA IN 36 SHELLFISH S AMPLES 。 S1=SAMPLE 1, S2=SAMPLE 2 , ETC.
