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Concentration calculation of Factor VIII for Elisa
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Nathan Coleman
Concentration calculation of Factor VIII for Elisa
My answer: 4.79 µg/ml
(All values below are µg/ml)
Calibration data
Determine the mean of the four absorbance values at each dilution.
The calibration curve is then a plot of concentration (x-axis) against mean absorbance. Note that the actual concentration in the assay is (2123/2200)/dilution where dilution is 20, 40 or 80. If you chart this you will see that the data fit well to a straight line.
Use the Excel SLOPE and INTERCEPT functions to derive the parameters of the trendline. (I get SLOPE = 30.59; INTERCEPT = -0.8225)
The equation for converting an observed absorbance value to a concentration is:
concentration = (absorbance - INTERCEPT)/SLOPE
Sample data
For the sample measurements the only one that is valid is that for the 20x dilution because the other absorbances lie outside the range of the calibration data (see note below).
Using 0.406 as the absorbance value in the equation above gives a value of 0.01596 for the concentration in the assay well. This sample was diluted 20-fold for the assay, and the source for that dilution was derived from a 15-fold ‘pre-dilution’ so the original concentration of the sample is 300x the assay value which is 4.788 µg/ml.
Note
I don't know the nature of the discrepancy between your calculations and the target value, but if you try to incorporate the 40x and 80x dilutions for the sample you will get higher values for the sample concentration. Presumably the assay becomes non-linear at low concentrations.
Determine the mean of the four absorbance values at each dilution.
The calibration curve is then a plot of concentration (x-axis) against mean absorbance. Note that the actual concentration in the assay is (2123/2200)/dilution where dilution is 20, 40 or 80. If you chart this you will see that the data fit well to a straight line.
Use the Excel SLOPE and INTERCEPT functions to derive the parameters of the trendline. (I get SLOPE = 30.59; INTERCEPT = -0.8225)
The equation for converting an observed absorbance value to a concentration is:
concentration = (absorbance - INTERCEPT)/SLOPE
Sample data
For the sample measurements the only one that is valid is that for the 20x dilution because the other absorbances lie outside the range of the calibration data (see note below).
Using 0.406 as the absorbance value in the equation above gives a value of 0.01596 for the concentration in the assay well. This sample was diluted 20-fold for the assay, and the source for that dilution was derived from a 15-fold ‘pre-dilution’ so the original concentration of the sample is 300x the assay value which is 4.788 µg/ml.
Note
I don't know the nature of the discrepancy between your calculations and the target value, but if you try to incorporate the 40x and 80x dilutions for the sample you will get higher values for the sample concentration. Presumably the assay becomes non-linear at low concentrations.
@Martin Not sure that I fully understand the numbers you mention, but if you have more than one assay of the sample that is in the range of the standard curve then you can simply use all valid readings to derive separate measurements of the [sample], remembering to vary the dilution factor as appropriate (e.g. x20 above but if the 40x dilution had also been valid then use the same formula but multiply by 600 (15x40) at the last step). You can then take the average value of all of your estimates of [sample]. (Apologies for using 'mean' and 'average' interchangeably here.)More
Hi Alan, Thank you´so much for the response. It really helped a lot and I have been able to work out how to perform this when only one of the samples are in range. Now I´m stuck instead on how to incorporate the values when all of them are in range. Example: Predilution for standard = 1, conc = 1,04 Avg ystd = 1,1585 0,611 0,3205 Y sample = 1,16 0,687 0,345 with a dilution of 30. I try to put them on a common slope, get a value for the relative potency and then calculate the concentration. The problem is that I get a value of 31,88 when the "correct" result is 33,50.More
(All values below are µg/ml)
Calibration data
Determine the mean of the four absorbance values at each dilution.
The calibration curve is then a plot of concentration (x-axis) against mean absorbance. Note that the actual concentration in the assay is (2123/2200)/dilution where dilution is 20, 40 or 80. If you chart this you will see that the data fit well to a straight line.
Use the Excel SLOPE and INTERCEPT functions to derive the parameters of the trendline. (I get SLOPE = 30.59; INTERCEPT = -0.8225)
The equation for converting an observed absorbance value to a concentration is:
concentration = (absorbance - INTERCEPT)/SLOPE
Sample data
For the sample measurements the only one that is valid is that for the 20x dilution because the other absorbances lie outside the range of the calibration data (see note below).
Using 0.406 as the absorbance value in the equation above gives a value of 0.01596 for the concentration in the assay well. This sample was diluted 20-fold for the assay, and the source for that dilution was derived from a 15-fold ‘pre-dilution’ so the original concentration of the sample is 300x the assay value which is 4.788 µg/ml.
Note
I don't know the nature of the discrepancy between your calculations and the target value, but if you try to incorporate the 40x and 80x dilutions for the sample you will get higher values for the sample concentration. Presumably the assay becomes non-linear at low concentrations.
(All values below are µg/ml)
Calibration data
Determine the mean of the four absorbance values at each dilution.
The calibration curve is then a plot of concentration (x-axis) against mean absorbance. Note that the actual concentration in the assay is (2123/2200)/dilution where dilution is 20, 40 or 80. If you chart this you will see that the data fit well to a straight line.
Use the Excel SLOPE and INTERCEPT functions to derive the parameters of the trendline. (I get SLOPE = 30.59; INTERCEPT = -0.8225)
The equation for converting an observed absorbance value to a concentration is:
concentration = (absorbance - INTERCEPT)/SLOPE
Sample data
For the sample measurements the only one that is valid is that for the 20x dilution because the other absorbances lie outside the range of the calibration data (see note below).
Using 0.406 as the absorbance value in the equation above gives a value of 0.01596 for the concentration in the assay well. This sample was diluted 20-fold for the assay, and the source for that dilution was derived from a 15-fold ‘pre-dilution’ so the original concentration of the sample is 300x the assay value which is 4.788 µg/ml.
Note
I don't know the nature of the discrepancy between your calculations and the target value, but if you try to incorporate the 40x and 80x dilutions for the sample you will get higher values for the sample concentration. Presumably the assay becomes non-linear at low concentrations.
More
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