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EIA Analysis Tools

This is the Cayman Chemical Company Enzyme Immunoassay (EIA) Tools page. It contains information, Excel workbooks, and other tools useful for anyone performing enzyme immunoassays.

If you have questions on the use of our tools, or feel something is missing, please contact Technical Services.

The Workbooks

Currently, Cayman Chemical uses two Excel workbooks to perform EIA analysis. Both are available for your download and use.

Download

Your downloading these files implies acceptance of the license agreement listed below and present in each workbook.

EIATriple ~ 650 Kb XLS formatted for assays performed in triplicate
EIADouble ~ 650 Kb XLS formatted for assays performed in duplicate

Trouble downloading? Right-click the link and select "Save Target As..."

These instructions are repeated on the first sheet of each workbook.

EIATriple Worksheet

EIADouble worksheet

Definition of Key Terms

Term	Definition
Blank	Background absorbance caused by Ellman's Reagent
TA (Total Activity)	Total enzymatic activity of the AChE-linked tracer
NSB (Non-Specific Binding)	Non-immunological binding of the tracer to the well
B₀ (Maximum Binding)	Maximum amount of tracer bound by antiserum in the absence of free analyte
Std0# (Standards)	Standards #1 through #8 made by serially diluting Std01 seven times. See diagram (concentrations may vary depending on the assay) Standards serial dilution
Smpl# (1:#)	Samples and their associated dilution factors [Dilutions must be entered in the format shown]
Ctrl# (1:#)	Positive controls and their dilutions (Cayman Chemical uses 1:5 dilutions of Std01 and Std03)
Assay	Pop-up menu to choose the assay you are analyzing. Selecting an assay inserts the value of Std01 and its units into cells B70 and H69, respectively
Stats	'y-int', 'slope' and R² are obtained from the log-regression fit of the calibration data. These values are automatically calculated and inserted into cells C79 through C81

Workbook Sheets

Analysis

Field	Explanation
LAYOUT	Contains sample names and associated dilution factors
RAW DATA	Data entered into the worksheet in the format defined by the LAYOUT section
SUBTRACTED	The AvgRawBlnk is subtracted from each cell
B/B₀	The value of each cell is computed by subtracting the AvgNSB and AvgBlank from the SUBTRACTED value, and then dividing by the AvgBo. The result of each cell is evaluated to determine if it is within the 20-80% B/B₀ linear range of the calibration curve, and highlighted accordingly
LOGIT	The logit for each cell is computed using the forumula: ln(B/B₀/((1 - B/B₀)). This technique "linearizes" the values to facilitate regression analysis
CONC	The concentration for each cell is calculated based on the LOGIT value, constants from the log-regression curve (see StdCurve section), and the dilution factor from the LAYOUT section
STANDARDS	The Avg B/B₀ Logit and Recalc values are calculated for each concentration value. Only the value of Std01 must be entered; subsequent values are calculated automatically. Recalc are the standard concentration values calculated from the regression constants
Samples	This table is compiled from the preceding sections. It includes Conc: average of the replicate concentrations from the CONCN section. Data outside the 20-80% B/B₀ range are highlighted %B/B₀: average of the replicate values from the B/B₀ section expressed as a percentage Dilution: the dilution factor, for that sample, from the LAYOUT section StdDev: the standard deviation of the replicate concentration values %CV: the coefficient of variance of the replicate concentration values SEM: the standard error of the mean for the replicate concentration values
STATS	y-int and slope are obtained from the log-regression fit of the calibration data. These values must be manuallytransferred from the StdCurve worksheet
CURVE	The IC20, IC50, and IC80 values are calculated. The IC20 and IC80 are the upper and lower detection limits of the standard curve, respectively. The IC50 is a quality control parameter (given as 50% B/B₀ in the kit booklet)
% Error	The absolute percent error of the control concentrations from their predicted value

StdCurve

This is the calibration curve of the concentration standards plotted versus the logit(B/B₀) values. The data are fit to a logarithmic regression line with the general equation: y = m ln(x) + b. The regression equation is displayed along with the correlation coefficient.

Instructions

The worksheets were designed to be as "hands-off" as possible. The following instructions assume you have loaded your plate according to one of the plate layouts shown above, and have the data in a form that can be imported (or pasted) directly into a spreadsheet. To analyze your data:

Choose the assay you wish to analyze with the pop-up menu
Input your sample names into the appropriate cells
Input the sample dilutions into the appropriate cells in the format shown (e.g., 1:5)
Paste/type your data into the RAW DATA section (starting in cell B12) of the Analysis worksheet. Select the "Values" option in the "Paste Special..." dialog box to maintain formatting

All concentration values, and relevant statistics, are automatically calculated. The Samples table is then filled in with the data. All averages and statistics are calculated based on all replicates for each sample. It is the responsibility of the individual researcher to decide if any data can be eliminated. Caution should be exercised, however, when doing this! Cayman Chemical has examined statistical methods to test for the presence of outliers in EIA assays. We find that individual replicates are rarely flagged as outliers, and cannot be eliminated from the experiment.

Note The sole purpose of the data contained in this workbook is to demonstrate the functions of the individual spreadsheets. It should not be used to determine any experimental parameters (e.g., standard dilutions, etc.).

Copyright and Disclaimer

Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:

Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.
Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.

THIS SOFTWARE IS PROVIDED BY CAYMAN CHEMICAL COMPANY, INC. "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL CAYMAN CHEMICAL BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

The views and conclusions contained in the software and documentation are those of the authors and should not be interpreted as representing official policies, either expressed or implied, of Cayman Chemical Company, Inc.

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Cayman Chemical Company · 1180 East Ellsworth Road · Ann Arbor, Michigan 48108 · USA

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