德国IBL ASP ELISA试剂盒使用说明（五）
b) Developing and reading the microplate strips
11. Carefully remove the plate sealer. Wash the wells 4 times with 300 μL Washing
buffer per well.
12. Add 100 μL of TMB peroxidase substrate (vial G) to all wells. Incubate at room
temperature (20-25°C) for 15 minutes. Protect from light.
13. Stop the reaction by adding 100 μL 0.3 M H2SO4 to all wells.
14. After 2-5 minutes, read the absorbance in a microplate spectrophotometer
using a 450 nm filter.
L . CALCULATION OF RESULTS
a) Calibration using the four-parameter logistic curve fit model
When the measured absorbance values of the standard dilutions are plotted on a
linear scale (y axis) against the DA-concentrations of the standard dilutions on a
logarithmic scale (x axis), a sigmoid (S-shaped) curve is obtained (see Fig. 5).
The non-linear 4-parameter logistic curve-fit model is extensively used for sigmoid
curves, in order to get accurate quantification of samples and a good fit at the
extremes of the curve. The following equation is given for a 4-parameter fitted
curve:
y = (a-d)/[1+(x/c)b]+d
where:
x is the concentration of DA in the standard/sample
y is the absorbance of the standard/sample
a is the y-value of the upper asymptote (Amax)
b is the relative slope of the curve at its center
c is the x-value at the midpoint of the curve (I50)
d is the y-value of the lower asymptote (Blank/Amin)
F I G U R E 5 .
NON-LINEAR CALIBRA-TION CURVE PREPARED BY 4-PARAMETER LO-GISTIC CURVE FIT .
b) Calculation formula
The following formula is used to convert ELISA results in pg/mL to shellfish
concentrations in mg/kg:
mg DA/kg = μg DA/g = 1 000 000 pg DA/mL · D · V / M
where:
pg DA/mL is the concentration of DA in the diluted extract
D is the dilution factor of the diluted extract
V is the volume of the methanolic extract (16 mL plus 4 g of homogenate giving nominal
20 mL total volume).
M is the mass of the shellfish homogenate (4 g).
c) Excel macro EMA31 calculation of DA concentration in shellfish samples
For calculation of assay results, a spreadsheet has been developed implementing
the calibration function and the conversion formula from pg/mL in the extract to
mg DA/kg shellfish.
1. Open the provided Excel Macro EMA31, enable macros and install the Solver
as described in the “Instructions” sheet of the Macro.
2. Copy the measured absorbance values (to at least 3 significant figures, e.g.
0.682) from the plate reading software result/report sheet and paste the values
in the Excel Macro EMA31 “Data Entry” sheet.
3. Enter the correct dilution factor used for the samples, in the corresponding
duplicate well windows.
4. Run the macro according to the instructions.
5. Go to the “Results” sheet. The results from the column “Shellfish sample DA
eqv. (mg/kg)” give the concentration of DA in the shellfish samples.
6. Sample concentrations should only be calculated from datapoints that are
within the valid working range of the standard curve as defined by the Excel
macro. If more than one sample dilution hit the working range of the standard
curve, we recommend that the dilution closest to the I50 value of the standard
is used.
Alternatively; another data analysis software (e.g. the software provided with
the plate reader) may be used as long as it supports the 4-parameter logistic
curve fit model.
d) Excel macro EMA31 calculation of DA concentration in Algal samples
1. Use the provided Excel macro EMA31 as described in the previous section.
2. Enter the correct dilution factor used for the algal samples, in the corresponding
duplicate well windows.
3. The results from the column “Sample extract/solution (pg/mL)” will provide the DA
concentration of the algae extracts as pg/mL.
4. If Pseudo-nitzschia cell counts are available for the filtered water sample, the
results can be converted to pg DA/cell, taking into account the volume of water
filtered and the extraction volume.
e) Excel macro calculation of DA concentration in seawater samples
1. Use the provided Excel macro EMA31 as described in the previous section.
2. Enter the correct dilution factor used for the seawater samples, in the
corresponding duplicate well windows.
3. The results from the column “Sample extract/solution (pg/mL)” will provide the DA
concentration of the seawater samples as pg/mL.
