ELISA Calculator Instructions

The Excel add-in can be downloaded from the Krishgen Online ELISA Calculator Page. To install the add-in, follow the procedure below:

After downloading you can copy the add-in somewhere on your hard drive or network. Use a  location which is writeable. As an example, we made a folder C:\

add-in and copied the KrishgenAddin.xlam to this location.

You may need to set the security settings for Excel for this folder to trust the folder.

To install, open excel and select options from the menu on Windows. On Mac OS select Excel add-ins from the Tools menu. You can also check this link https://support.office.com/en-us/article/Add-or-remove-add-ins-in- Excel-0af570c4-5cf3-4fa9-9b88-403625a0b460#OfficeVersion=Windows

In Windows follow the following menus Click Add-ins. Press the go button in the bottom of the windows Then click browse and select the add-in.
Once it is installed a Menu named KRISHGEN appears.

If you click the menu all Krishgen Online ELISA Calculator buttons are visible.
By clicking the right ELISA button, a curve fitting sheet will appear with experimental data.

In the top table called INPUT, you can paste your data in 96-well format.

In the second table, you can identify where in the 96 well plate calibrators, controls, blanks and samples are located.
This can be performed by typing or copying the identifies in the table or by using the plate layout builder.

Assigning calibrator, sample control and blank positions

Krishgen Online ELISA Calculator version 1.3 and higher has an option to create a plate layout to identify sample positions.

In the paragraph above, an ELISA sample curve fitting sheet is shown. Some identifiers contain the suffix CAL-. This indicates the position in the plate is a calibrator. The concentration of the calibrator is indicated after CAL-. CAL-500 means a calibrator with concentration 500. The layout may contain more than one CAL-500 and the program will calculate the average of the corresponding input values and copy this average to the input section.

The CAL-500 replicates may be anywhere on the plate not necessarily next to each other. You can also add other sample ID information in the layout field for your information. The sample ID information will not be sent over the internet.

Calibrator values can be entered manually or using copy-paste, or you can use the layout builder to enter these. By clicking the layout builder from the Krishgen Online ELISA Calculator menu a form will appear with buttons.

By clicking the Sample -> and Replicate -> the fill direction in the layout of the sample numbers and replicates can be assigned. Also, the number of replicates can be defined.

Then select the region in the sample ID table you wish to assign as a calibrator.

If you then click on the Cal button of the Layout builder. The software will ask you for the highest concentration of the calibrator and the dilution factor. Suppose we would type in 500 and 2 respectively then the software will fill the region starting with replicates of the highest calibrator (500) and then replicates of a 2x diluted calibrator = CAL-250 and continue for each dilution until  the selected region is filled.

You can use the layout builder also to add Blank, control and sample IDs into the table to identify their location.

The “CAL-“ identifier before the value can be removed by clicking the toggle calibrators button. When a cell contains a number representing the calibrator concentration, a calibrator can be assigned by selecting this cell or cells and clicking the toggle calibrators button. It will then place “CAL-“ before the value.

Dilutionfactors

Usually, samples are diluted when they are measured. The dilution can be indicated in the dilution table.

Dilutions can be copied or typed into this table or alternatively the layout builder can be used to assign a dilution factor to certain cells.

Copy the values of the standards to the calibration input table

By clicking on the Calibrators from layout button the concentration of the calibrators and the average calibrator values from the corresponding input field will be copied to the input field. The input field contains values needed to perform curve fitting and calculate unknown values. The input columns can be found from column “AL” in the worksheet.

Assigning weights to the calibrators
If you like to use weights in the curve fitting, you can apply weights by hitting the weights button. This button will copy weight formulas to the weight column. In this case it will use 1/(signal)2 By clicking this button again, the weight formulas will disappear again.

Alternative weight calculation can be applied by entering any formula or number in the weights column before curve fitting.
You can now perform curve fitting in the template or online by hitting the Fit Current Sheet button.

A form will appear in which you can enter several fit settings.

The fit function is the function to which the calibrator data points will be fitted. A good start is a 4PL fit.

The email field is only for information, but you can leave this blank or type in any name you like. The key in this example is entered as “free” and this this will enable you to perform 10-15 free curve fits per day.

In the “Fit current sheet” form, can also select a server close to you for improved speed. There are also offline options.

When hitting the perform fit button the values in the input field will be used to fit a calibration curve. This calibration curve will be used to backfit all the values in the 96 well plate. The calculated concentration will be shown in a table. Also, a concentration x dilution table will be shown.

A report will also be generated on the right of these tables. The report contains the calibrator values with the fitted curve. The fit function and the values of the fit parameters (A, B, C, D). It will also show goodness of fit parameters R2 and sum of squares (SSQ).

The replicate signal values of the samples will be shown as well as the calculated concentration values with average and CV%.

DATA IN AN X-Y FORMAT:

Krishgen’s ELISA Calculator also has other options for curve fitting than the frequently used 96 well format.

Sometimes the data is presented as x and y and the values are not in 96 well format, but just in one column.

For this you can use XY or XY+ button in the Krishgen ELISA Calculator menu.

Using these options, you can directly enter the standard curve data points in the x-data and y-data columns. The unknown samples can be entered into the y-known column.

DATA IN ANY OTHER FORMAT

By clicking the Raw button, a raw example appears as an example of an output file from an assay reader. The data can be anywhere and can have any format, not just 96 wells.

The top example has a top to bottom calibration, the bottom a left to right calibration. The add- in can work both with left to right and top to bottom layouts.

With the Fit input ranges button you can select regions which will be used for the curve-fitting

A window will appear. Selecting the icon on the right of the input boxes will display a select box.  Selecting a region will then copy the location of this region into the field.

You can also select the regions from left to right as is indicated in the following example.

By clicking on the Absolute click box the selection will make the reference absolute. This will allow to enter values from another sheet than the active sheet.

With the buttons Clear input, you can clear the input ranges.

With the save range button you can save the regions on your computer. This is however usually done automatically. The settings will be loaded every time you open the window.

With save settings you can save your username, key, server and fit function on your computer.