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The calibration curve concentrations used for the ELISA's were 300 U/L, 150 U/L, 75 U/L, 37.5 U/L, 18.75 U/L, 9.38 U/L, 4.69 U/L.
Average the duplicate readings for each calibrator, control, and samples and subtract the average zero calibrator optical density. Create a calibration curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a calibration curve by plotting the mean absorbance for each calibrator on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the LDHA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the calibration curve must be multiplied by the dilution factor.