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Posted by star on 2017-12-29 17:07:54 Hits:32
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In this article, we will demonstrate one way of analyzing some typical ELISA data using GraphPad Prism. Prism have been one popular software for a few years now, and it does a very good job of simplifying data analysis. If you have never used Prism before, I would definitely encourage you to try it. Before analyze ELISA data with Graphpad Prism, you need to organize our data in Excel, so that it will be easy to copy into Prism for analysis. Prism does have tools of its own that can be used to manipulate your raw data, but my personal preference is to have the data in the Excel format before bringing it into Prism.

So for typical ELISA data, you will want to have three columns. The column first will contain the known concentration of your the standards, and their units and value depends completely on how you prepared your standard samples. The second two columns will contain the absorbance measurements for each of the standard curves.Before the absorbance measurements of the standards,you need to place the absorbance measurements of your samples. Because you measured samples in duplicate, the duplicate measurements must be side-by-side one another, and they will automatically be averaged by Prism later on. If you only had one absorbance measurement per samples. You would leave the right column for these samples empty. If the samples were measuredin triplicate, then you would have three columns, one for each measurement. In any case,the concentration column should always be empty for the samples since you dont know their concentration yet.

Now, you will copy the data and open up Prism. For those of you who are new to Prism, the first thing that we must do is select our desired type of Table and Graph. You can seeto the left that Prism has a number of different Table and Graph types to choose from, and choosing the correct table type to match data. For ours tandard curve data, we will use an X-Y Table. As for these other options, your selection should be based on the maximum number of absorbance measurements you have for your standards and samples. You will choose to "Enter 2 replicatevalues in side-by-side subcolumns," if you have 2 replicate values (or, duplicates) for your standard curves. If you had three standard curves, or made triplicate measurements of your samples, you would want to change this number to a three, and so forth.

Now youll click Create. You can see on the left that you now have an empty Data table, called Data 1, and a Graph which is also called Data 1. In Prism, data tables and graphs are linked to one another, so any changes made to a Table will automatically be reflected in its corresponding Graph. You can see that the Graph is empty because you havent put any data into your table yet.
Now we will paste our standard curve data into the table. Lets label this column "Concentration (ng/mL)", and this one "Absorbance (450 nm)."Some of you may be working with different units of concentration, or even log-concentrations,so simply label the columns accordingly. Prism will ask us here to choose how you want your graph to look. You can select the plot with no connecting lines, because connecting lines would obscure the curve that you will fit to this data in the next step. You can see now that the Standard Curve data is here on your graph. Also notice that the labels that we added to our data table are automatically added to our Graphas axis titles. You can zoom in a little bit to see things more clearly. Notice to the right, there is a button called "Interpolate a standard curve." The dialog box that opens gives you a number of choices, and each one may be appropriate depending on the shape standard curve data. If you click on one of the choices, the Details button appears. Clicking on that reveals the shape of the curve that will be fit using that selection, as well as some advanced options and settings, which you will not get into for the sake of time. But information about these and all other aspects of Prism, and data analysis in general, can be found in the Prism helpguide which can be accessed via button with a question mark. Back to curve selection, You will find thata "Second Order Polynomial" is a good choice for ELISA standard curve data. You will leave the Outliers options set to the default of "No special handling of outliers," and check the "Report each interpolated value option. Here, you can tell Prism to calculatea confidence interval for the interpolateddata, but this is optional so you will not do it here. You will also check the "Plot curve"option and leave it set to the 95% confidenceband, so that you can examine how well the curve fits the data. Hit OK. You can see to the left that Prism has createda new page in the "Results" folder, labeled "Interpolation of Data1," and this has two subpages.

As I said before, the first thing we should do is check that the curve we created fits our data well. At first glance, you can see that the trend line fits the higher concentrations in our standard curve well, but you cant see the lower concentrationsvery easily. You can first duplicate this graph by right-clicking on it and selecting "Duplicate Current Sheet." Now, you will change the range of the X and Y axes of the new graph by double-clickingon them. You will divide the maximum x and y values by four in order to better visualize those smaller concentrations.
 
Now you can see clearly that the curve fits the standard curve data well, so now you can retrieve your interpolated concentration data from the Results that were generated. Clickon "Interpolated X mean values." Here, you can see the absorbance measurements of your unknown samples, and to the left of them are their Interpolated Concentrations. If you diluted your samples before adding them to the ELISA plate, then you will need to multiply these concentrations by whatever the dilution factor was. For example, if you dilute the samples 10-fold, then their actual concentration would be ten times higher than those shown here.

So there you have it! You have now determined the concentration of your unknown samples by interpolating your standard curve data with Prism. The next step will be for you to organize your data appropriately, graph it and do statistical analysis for your next paper or presentation. 
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