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EIA Analysis Tools
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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
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Term
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Definition
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Blank
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Background absorbance caused by Ellman's Reagent
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TA (Total Activity)
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Total enzymatic activity of the AChE-linked tracer
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NSB (Non-Specific Binding)
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Non-immunological binding of the tracer to the well
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B0 (Maximum Binding)
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Maximum amount of tracer bound by antiserum in the absence of free analyte
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Std0# (Standards)
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Standards #1 through #8 made by serially diluting Std01 seven times. See diagram (concentrations may vary depending on the assay)
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Standards serial dilution
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Smpl# (1:#)
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Samples and their associated dilution factors [Dilutions must be entered in the format shown]
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Ctrl# (1:#)
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Positive controls and their dilutions (Cayman Chemical uses 1:5 dilutions of Std01 and Std03)
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Assay
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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
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Stats
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'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
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Workbook Sheets
Analysis
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LAYOUT
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Contains sample names and associated dilution factors
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RAW DATA
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Data entered into the worksheet in the format defined by the LAYOUT section
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SUBTRACTED
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The AvgRawBlnk is subtracted from each cell
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B/B0
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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
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LOGIT
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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
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CONC
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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
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STANDARDS
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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
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Samples
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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
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STATS
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y-int and slope are obtained
from the log-regression fit of the calibration
data. These values must be manually
transferred from the StdCurve worksheet
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CURVE
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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)
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% Error
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The absolute percent error of the control concentrations from their predicted value
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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
Copyright © 2006 Cayman Chemical Company, Inc. All rights reserved.
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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