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Click “legend” tag and uncheck “show legend” 7.Click “Title and label” tag, and write the graph title, names of X and Y axis. 6.Indicate the column of Ln(⊿Blank) (in our example G2-G8) as date area of X, then “next.” 5.Click “series” and choose data area of X. 4.Indicate the column of “Ln(conc)” (in our example, B2-B8 as date area. 3.Choose default, i.e.” plotting only” then next. Preparation of reversed graph and regression equation Then transform ΔBlk (F column) in the same way to fill column G.īy using this table, prepare “a reverse standard curve for calculation” with following steps. In B2 cell, write” =LN(A2) “ (do not include quotation marks), the logarithmic value will appear in B2, and using fill-handle, transform other concentrations into (B3-B8) Subtract the mean of blank absorption from each mean absorbance to make ΔBlank.Īdd two columns for logarithmic transformation.įor transformation, natural logarithm is more convenient. ![]() If possible, as absorbance, difference of absorbance at 450nm and 620nm is preferable. The example shown here is a duplicate assay, and as TMB is used as chromogenic substrate, we measured absorbance at 450nm. Standard points of rat insulin: 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10.0 ng/mlįirst make up a table for standard concentration and absorbance as shown below. Procedure of calculation step-by-step with an example of our insulin assay data Input of data in EXCEL spread sheet. The procedure will be shown step by step.Īs in ELISA, the standard curve is nearly linear and excellent fitness is easily obtained by logarithmic transformation of both absorbance and concentration, the method starts from logarithmic transformation of the data. I suggest a method to calculate assay value by using a reverse standard curve where absorbance on X and concentration on Y. so, the usual standard curve by EXCEL is not useful for assay value calculation. ![]() In usual step for calculation of the assay value of ELISA is to draw a standard curve, absorbance on Y-axis against concentration on X-axis, then to estimate assay value from the absorbance of the sample.ĮXCEL is really an excellent tool, however, it does not give X value from Y. 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