Let’s check fitness of the equation by calculation the assay value from standard data. The R2 value, 0.9998, obtained indicates that the fitness of the third order regression curve in our example seems to be excellent. From R2, we can estimate fitness of the curve. Now, we get the reverse standard curve with equation and R2. Click “option” tag, and check “show equation” and “show R-2”., then “OK”. 10.Choose “multimember regression”, and set the order “3”. 9.Click one data point on the graph to make color change, and click “graph” in the task bar, then choose “Add regression curve” Y-axis is move in the similar way to X-axis. To move X axis, double click the figure on X-axis to show X-axis setting window, and at “scale” tag uncheck the automatic checking of “intersection with Y”, and write the intersection wanted (in our example,” -5”, then OK. 8.Click ”finish” to show a graph on the data sheet.Īs the graph appears as above is not good looking, so we should move X and Y axis. 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. As for these samples, the concentration obtained from the standard curve must be multiplied by the dilution factor when analyzing the results.Technical Consultant, Shibayagi Co., Ltd. If the OD value of the sample is out of upper limit of the standard curve in sandwich(or out of the lower limit of competitive ELISA), these samples should be diluted before proceeding with the ELISA to obtain an accurate result. Select the 4th work sheet (Fit NLF Find X from Y1), input or copy the OD value of the sample to the left column, the concentration will be calculated automatically. Step 6: Calculate the concentration according to the curve Then double click the X axis and Y axis to set them to log type. Step4: Check the information of the standard curve (parameter values, R-Square, etc.)ĭouble click the Fitted Curve Plot in step 4 to make the curve to editable state. Select the value box, and then select items in turn as below.Īfter setting the above items, click the Fit button, then the curve and its information is produced as showed in step 4. Step 1: Input information of the OD value and concentration of the standards The software Origin is recommended to use. Plot a four parameter logistic curve on log-log graph paper, with standard concentration on the x-axis and OD values on the y-axis. Subtract the average value of zero standard OD (this step is unnecessary in procedure of competitive ELISA). ELISA samples are always tested in duplicates or in triplicates, then average the absorbance values for each setĢ. As the last procedure of ELISA, calculation of results is of great importance to the success of the assay.ġ.