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.