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The calibration curve concentrations used for the ELISA's were 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.12 ng/mL.
Average the duplicate readings for each calibrator, control, and samples and subtract the average zero calibrator optical density. Create a calibration curve on log-log graph paper, with APOD concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the calibrator points and it can be determined by regression analysis. Using some plot software is also recommended. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.