ELISA

Development
Guide

a guide
for the
use of
antibodies
in ELISA
development

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Calculation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Assay Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-8
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8-12
Technical Service Troubleshooting Questionnaire . . . . . . . . . . . . . .13-15
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

The ELISA Protocol, as well as the guidelines and tips for

building your own ELISA, are based on using R&D
Systems’ antibody pairs tested for ELISA.

Distributed by:
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United Kingdom
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Germany
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Borsigstrasse 7 Fax: +49 (0)6122 909819
65205 Wiesbaden-Nordenstadt E-mail: infogmbh@RnDSystems.co.uk
2

Introduction
The ELISA (Enzyme Linked Immunosorbent Assay) technique is based on the antibody
sandwich principle. First, a capture antibody specific to the analyte of interest is bound to a
microtiter plate to create the solid phase. Unbound antibody is removed by washing the
plate and a blocking reagent is added. Following a wash, samples, standards, and controls
are then incubated with the solid phase antibody, which captures the analyte. After
washing away unbound analyte, a conjugated detection antibody (e.g. biotin conjugated)
is added. This detection antibody binds to a different epitope of the molecule being
measured, completing the sandwich. Following a wash to remove unbound detection
antibody, a detection reagent (e.g. streptavidin-HRP) is added. The plate is washed, a
substrate solution (e.g. TMB/hydrogen peroxide) is added and color develops in proportion
to the amount of bound analyte. Color development is stopped and the intensity of the
color is measured.

The assay in Figure 1 involves a detection system which utilizes streptavidin-HRP (R&D
Systems, Cat. # DY998) and tetramethylbenzidine (TMB)/peroxide (R&D Systems,
Cat. # DY999) as a substrate. TMB/peroxide turns blue when modified by HRP. The final step
is to stop the reaction with an acidic solution, which turns the solution yellow. The optical
density (O.D.) of the yellow color is read at A450 on a microtiter plate reader.
Figure 1.

B B B
B

Step 1. Analyte-specific antibody (capture Step 2. A biotin-labeled analyte-specific detec-

antibody) is pre-coated onto a microplate. tion antibody binds to a second epitope on the
The sample is added and any analyte present analyte forming the analyte-antibody complex.
is bound by the immobilized antibody.
Yellow
Color

Blue
Color

H
SA Stop Solution
H
SA H H H
SA SA SA

B B B B B B
H
SA Substrate

Step 3. Streptavidin-HRP is added and binds Step 4. TMB/peroxide (substrate) is added and
to the biotin-labeled detection antibody. converted by the HRP (enzyme) to a color
Legend product (blue) in proportion to the amount of
Sample Antigen Capture Antibody analyte bound (signal increases as analyte
concentration increases). The reaction is
B
stopped upon addition of stop solution,
Antibody-Biotin Conjugate H Horseradish Peroxidase
SA
Conjugated Streptavidin changing the solution from blue to yellow.

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 3

Supplies
Materials required but not supplied
• ELISA microtiter plates (Costar, Catalog # 2592 or equivalent)
• Disposable plate sealers (Costar, Catalog # 3095 or equivalent)
• Disposable reagent reservoirs (Baxter, Catalog # 5082-128 or equivalent)
• Assorted graduated cylinders
• Wash bottle and/or automatic plate washer
• Assorted adjustable volume pipettes
• 8 or 12 channel multichannel pipettes
• Pipette tips
• Assorted volume pipettes
• Polypropylene tubes
• ELISA plate reader with optional data reduction software

Solutions required but not supplied

• Wash Buffer - 0.05% Tween 20 in PBS, pH 7.4
• Diluent - refer to the ELISA Protocol on the antibody insert for exact formulation as important
differences occur
• Detection System - e.g. streptavidin-HRP (R&D Systems, Catalog # DY998), Color Reagent A (H202)
and Color Reagent B (TMB) (R&D Systems, Catalog # DY999)
• Stop Solution - based on detection system

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4

ELISA Protocol
Plate Preparation
1. Transfer 100 µL/well of the capture antibody (diluted to the appropriate concentration
in PBS, use immediately) to an ELISA plate. Seal plate and incubate overnight at room
temperature.
2. Aspirate each well and wash with Wash Buffer, repeating the process for a minimum of
3 washes. Wash by forcefully filling each well with Wash Buffer (400 µL) using a squirt
bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of
liquid at each step is essential to good performance. After the last wash, remove any
remaining Wash Buffer by aspirating and by inverting the plate and blotting it against
clean paper toweling.
3. Block plates by adding 300 µL of recommended Blocking Buffer (see package insert) to
each well. Incubate at room temperature for a minimum of 1 hour.
4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Alternatively, the blocking buffer can be aspirated after step 3 and the plates can be
dried under vacuum. When sealed with desiccant, the plates can be stored at 4°-8° C for
at least 2 months.

Assay Procedure
1. Dilutions of unknowns and standards should be carried out in polypropylene tubes. Add
100 µL of sample or standards in an appropriate diluent, per well. Mix by gently tapping
the plate frame for 1 minute. Cover with an adhesive strip and incubate 2 hours at room
temperature.
2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
3. Add 100 µL of the biotinylated detection antibody, diluted in appropriate diluent, to
each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
5. Add 100 µL Streptavidin-HRP (R&D Systems, Catalog # DY998, dilute according to the
directions on the vial label) to each well. Cover the plate and incubate for 20 minutes at
room temperature. An alternate detection system may be used. Avoid placing the plate
in direct light.
6. Repeat the aspiration/wash as in step 2 of Plate Preparation.
7. Add 100 µL Substrate Solution (R&D Systems, Catalog # DY999) to each well. Incubate for
20 - 30 minutes at room temperature. Avoid placing the plate in direct light.
8. Add 50 µL Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
9. Determine the optical density (O.D.) of each well within 30 minutes. If using
R&D Systems Catalog # DY999 or TMB, set the microtiter plate reader to 450 nm. If
wavelength correction is available, set to 540 or 570 nm. If wavelength correction is not
available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This
subtraction will correct for optical imperfections in the plates. Readings made directly at
450 nm without correction may be higher and less accurate.

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 5

Calculation of Results
Manual
The values of the unknown samples are assigned in relation to the standard curve. If data
reduction software is not available, calculate assay results by averaging the duplicate
readings and subtracting the zero standard optical density (O.D.) from the sample O.D.
Construct a standard curve by plotting the standard O.D. points by hand and drawing a
best fit or point-to-point curve. Plotting the data using log/log or semi-log paper is
acceptable. If samples have been diluted, the concentration read from the standard curve
must be multiplied by the dilution factor. A standard curve should be generated for each
set of samples assayed.

Automated
The values of the unknown samples are assigned in relation to the standard curve. Most
labs have plate reader software or other software that allows various methods of curve
fitting to be tried. It is recommended that various methods (e.g., linear, semi-log, log/log,
4 parameter logistic) be tried to see which curve best fits the data. One way to determine if
the curve fit is correct is to backfit the standard curve O.D. values. To do this, first plot the
standard curve. Next, treat standards as unknowns and interpolate the O.D. values from
your standard curve. They should read close to the expected values (+/-10%). Use the data
reduction method that gives the best correlation (r) value and backfit.

Assay Optimization
Once an acceptable standard curve has been obtained using the recommended
protocol and reagent concentrations, optimize the assay to meet performance
requirements.

There are many parameters which influence the results obtained in an ELISA. These
include: antibody quality and concentrations, incubation times, incubation temperatures,
detection reagent quality and concentration, and substrate type and quality. For this sec-
tion, it is assumed that all recommended reagents are being used.

Antibody concentration - the best way to determine the optimal capture and detection
antibody concentrations is to perform a grid experiment. A grid experiment provides a
method to test many antibody pair concentrations using only one plate. Antibody starting
concentrations will vary depending on antibody type (monoclonal versus polyclonal) used
for capture and detection, see Table 1. Refer to the product inserts for capture and
detection antibody types as well as recommended starting concentrations.

Table 1. Recommended antibody starting concentrations

Monoclonal Capture/ Monoclonal Capture/ Polyclonal Capture/
Polyclonal Detection Monoclonal Detection Polyclonal Detection

Capture Concentration 1, 2, 4 and 8 µg/mL 0.5, 1, 2 and 4 µg/mL 0.2, 0.4 and 0.8 µg/mL

Detection Concentration 50, 100, 200 and 0.25, 0.5, 1 and 2 µg/mL 50, 100, 200 and
400 ng/mL 400 ng/mL

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6

Assay Optimization cont.

To form the grid, divide a 96-well plate into 4 quadrants. See Figure 2 for an example of a
monoclonal capture-polyclonal detection grid experiment. The 6 columns in each
quadrant represent capture antibody concentrations, the 4 rows in each quadrant
represent standard curve points, and each of the 4 quadrants represents a different
detection antibody concentration. Each quadrant is a "mini-grid", identifying different
capture antibody and standard concentrations at one particular detection antibody
concentration. In the grid experiment in Figure 2, each quadrant contains all the possible
combinations of capture antibody at 1, 2 and 4 µg/mL and standard curve points of ∅
(Diluent stated on the product insert), 1000, 2000, and 4000 pg/mL, at one detection
antibody concentration.

From the multiple combinations of antibody pair concentrations illustrated on the grid,
select the concentrations that give the best signal to noise ratio. The ∅ standard points
give the "noise" or the background value that can be expected at each of the antibody pair
concentrations. The 1000, 2000 and 4000 pg/mL standard curve points give the "signal"
resulting from each of the many antibody pair concentrations. Select the highest signal to
noise ratio that still gives an acceptable background. A signal to noise ratio of at least 10 is
excellent, but the ratio should be at least five.

Figure 2. Grid experiment for monoclonal capture-polyclonal detection assay

50 ng/mL detection 100 ng/mL detection

1 2 3 4 5 6 7 8 9 10 11 12

1 µg/mL 1 µg/mL 2 µg/mL 2 µg/mL 4 µg/mL 4 µg/mL 1 µg/mL 1 µg/mL 2 µg/mL 2 µg/mL 4 µg/mL 4 µg/mL
capture capture capture capture capture capture capture capture capture capture capture capture

A ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅

B 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
pg/mL pg/mL
standard standard

C 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000
pg/mL pg/mL
standard standard

D 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000
pg/mL pg/mL
standard standard

E ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅ ∅

F 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
pg/mL pg/mL
standard standard

G 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000
pg/mL pg/mL
standard standard

H 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000 4000
pg/mL pg/mL
standard standard

200 ng/mL detection 400 ng/mL detection

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 7

Assay Optimization cont.

Background - <0.2 O.D. units. Factors that influence background include: blocking reagent,
capture and detection antibody concentrations, detection system, incubation times, dilu-
ents, and washing technique.
Curve height - preferably above 1.0, usually between 1.0 and 3.0 O.D. units. Factors that
influence curve height include: capture and detection antibody concentrations (see grid
experiment in Figure 2), incubation times and temperatures, detection system concentra-
tion, avidity of antibodies for antigens, pH, diluents, and quality of reader.
Detection system - assay sensitivity may increase with increasing detection reagent con-
centration or alternate detection system. However, this may result in higher background
readings.
Dilution of serum and plasma samples - serum and plasma samples may require a dilu-
tion of at least 2-fold in an appropriate buffer to overcome matrix effects. Empirically
determine the dilution of the samples required to result in linearity of dilution. When dilut-
ing samples, remember that the diluent used for the standard curve should be the same as
that used for samples. If samples are diluted, include the appropriate dilution factor when
calculating results.
BSA - bovine serum albumin, used as a blocking and carrier protein. Since different grades
of BSA exist and may contribute to background, an ELISA grade BSA should be chosen and
validated.
Incubation temperatures - the sample and detection antibody incubations should be
performed at room temperature. Sample incubation overnight at 4° C or 1 hour at 37° C
may increase assay sensitivity, but may also increase the background.
Incubation times - sensitivity may be increased with a longer incubation time at room
temperature. Be aware that the top of the curve may flatten out and become unusable,
limiting the assay range. Additionally, background may increase.
Interfering substances - it is important to be aware of the possible presence of interfer-
ing substances such as heterophilic antibodies or rheumatoid factors. Please refer to The
Immunoassay Handbook, edited by David Wild, Nature Publishing Group, copyright 2001,
for suggestions on how to control these substances.
Reagent reconstitution and storage conditions - reconstitution and storage instructions
provided with each reagent must be followed to ensure proper reagent perfomance.
Sample preparation and storage - while not every analyte has the same stability within a
given matrix, there are general precautions which should be followed. Samples that are
not used immediately after preparation should be stored in single use aliquots at -70° C.
A -20° C freezer may be acceptable, depending on analyte, if it is a manual defrost freezer.
It is best if the samples contain carrier protein. Multiple freeze-thaw cycles should be
avoided.
Samples/standard volume - use of a larger sample/standard size (200 µL per well vs.
100 µL per well) may increase sensitivity.
Substrate - substrates can vary. However, choosing an alternate substrate will require addi-
tional assay condition optimization. Some substrates require a longer incubation time to
get the curve to a reasonable height. If the substrate is functioning as expected, sensitivity
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8

Assay Optimization cont.

may be enhanced by increasing incubation time. Monitor the plate as it is developing to
avoid excessively high backgrounds. Typically, the incubation time ranges from 10 to 30
minutes. Use the correct filters required to read the appropriate wavelength for the sub-
strate chosen. This information is available from the substrate vendor.
Use of a shaker - at room temperature may increase sensitivity. Shakers may be used for
some or all of the incubation steps. Incubation times would have to be determined empiri-
cally.
Washing - follow washing instructions given in the ELISA Protocol, page 4. Insufficient
washing can result in high coefficients of variation (CVs), high background, and poor
results.
Sensitivity - varies for each antibody pair. Sensitivity is defined by reliable discrimination
from the zero standard. Factors which influence sensitivity include: capture and detection
antibody concentrations (refer to the grid experiment shown in Figure 2), incubation times
and temperatures, avidity of antibodies for antigens, sample/standard volumes, pH, dilu-
ents and wash buffer formulation. However, there is a limit to the sensitivity that can be
achieved with each antibody pair.

Troubleshooting Guide
Problem Possible Cause Solution

High Background • Insufficient washing • See washing procedure on

page 4
• Increase number of washes
• Add a 30 second soak step
inbetween washes

• Too much streptavidin-HRP or • Check dilution, titrate if

equivalent necessary

• Insufficient blocking • Check blocking solution

calculations
• Increase blocking time

• Incubation times too long • Reduce incubation times

• Interfering substances in • Run appropriate controls

samples or standards

• Buffers contaminated • Make fresh buffers

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 9

Troubleshooting Guide cont.

Problem Possible Cause Solution

No Signal • Reagents added in incorrect • Repeat assay

order, or incorrectly prepared • Check calculations and make new
buffers, standards, etc.
• Review protocol

• Contamination of HRP with azide • Use fresh reagents

• Not enough antibody used • Increase concentration

• Standard has gone bad (if there • Check that standard was handled
is a signal in the sample wells) according to directions.
• Use new vial

• Buffer containing FCS used to • Requalify your reagents of choice

reconstitute antibodies

• Capture antibody did not bind to • Use an ELISA plate (not a tissue
plate culture plate)
• Dilute in PBS without additional
protein

• Buffers contaminated • Make fresh buffers

Too much signal - whole • Insufficient washing/washing • See washing procedure on

plate turned uniformly blue step skipped - unbound page 4
peroxidase remaining

• Substrate Solution mixed too • Substrate Solution should be

early and turned blue mixed and used immediately

• Too much streptavidin-HRP • Check dilution, titrate if

necessary

• Plate sealers or reagent • Use fresh plate sealer and

reservoirs reused, resulting in reagent reservoir for each step
presence of residual HRP. This
will turn the TMB blue non-
specifically

• Buffers contaminated with metals • Make fresh buffers

or HRP

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10

Troubleshooting Guide cont.

Problem Possible Cause Solution

Standard curve achieved • Not enough streptavidin-HRP • Check dilution, titrate if

but poor discrimination necessary
between points (low or flat
curve) • Capture antibody did not bind • Use an ELISA plate (not a tissue
well to plate culture plate)
• Dilute in PBS without additional
protein

• Not enough detection antibody • Check dilution, titrate if

necessary

• Plate not developed long enough • Increase Substrate Solution

incubation time
• Use recommended brand of
Substrate Solution

• Incorrect procedure • Go back to General ELISA

Protocol; eliminate modifica-
tions, if any

• Improper calculation of standard • Check calculations, make new

curve dilutions standard curve

Poor Duplicates • Insufficient washing • See washing procedures on

page 4
• If using an automatic plate
washer, check that all ports are
clean and free of obstructions,
add a 30 second soak step and
rotate plate halfway through the
wash

• Uneven plate coating due to • Dilute in PBS without additional

procedural error or poor plate protein
quality (can bind unevenly) • Check coating and blocking
volumes, times and method of
reagent addition. Check plate
used
• Use an ELISA plate (not a tissue
culture plate)

• Plate sealer reused • Use a fresh plate sealer for each

step

• No plate sealers used • Use plate sealers

• Buffers contaminated • Make fresh buffers

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 11

Troubleshooting Guide cont.

Problem Possible Cause Solution

Poor assay to assay • Insufficient washing • See washing procedure on

reproducibility page 4
• If using an automatic plate
washer, check that all ports are
clean and free of obstructions

• Variations in incubation • Adhere to recommended

temperature incubation temperature
• Avoid incubating plates in areas
where enviromental conditions
vary

• Variations in protocol • Adhere to the same protocol

from run to run

• Plate sealer reused, resulting in • Use fresh plate sealer for each
presence of residual HRP which step
will turn the TMB blue

• Improper calculation of standard • Check calculations, make new

curve dilutions standard curve
• Use internal controls

• Buffers contaminated • Make fresh buffers

No signal when a signal is • No cytokine in sample • Use internal controls

expected, but standard • Repeat experiment, reconsider
curve looks fine experimental parameters

• Sample matrix is masking • Dilute samples at least 1:2 in

detection appropriate diluent, or prefer-
ably, do a series of dilutions to
look at recovery

Samples are reading too • Samples contain cytokine levels • Dilute samples and run again
high, but standard curve above assay range
looks fine

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12

Troubleshooting Guide cont.

Problem Possible Cause Solution

Very low readings across • Incorrect wavelengths • Check filters/reader

the plate

• Insufficient development time • Increase development time

• Coated plates are old and have • Coat new plates

gone bad

• Capture antibody did not bind to • Use an ELISA plate (not a tissue
the plate culture plate)
• Dilute in PBS without additional
protein

• Buffer containing FCS used to • Requalify your reagents of choice

reconstitute antibodies

Green color develops upon • Reagents not mixed well enough • Tap plate
addition of stop solution in wells
when using streptavidin-
HRP

Edge Effects • Uneven temperatures around • Avoid incubating plates in areas

work surface where environmental conditions
vary
• Use plate sealers

Drift • Interrupted assay set-up • Assay set-up should be

continuous - have all standards
and samples prepared appropri-
ately before commencement of
the assay

• Reagents not at room • Ensure that all reagents are at

temperature room temperature before
pipetting into the wells unless
otherwise instructed in the
antibody inserts

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 13

Technical Services Troubleshooting

Questionnaire
Please consult the ELISA Development Guide to help resolve a problem before submitting
this form. Fax the completed Troubleshooting Information Sheets to Technical Services at
612-379-6580 in the United States or +44 (0)1235 551129 in the United Kingdom.

Products Used (fill in the blank spaces)

Plates
Manufacturer:_____________________________________________________________
Type: ____________________________________________________________________

Blocking Buffer
Buffer components: ________________________________________________________
Date made: _______________________________________________________________
Time/Temperature:_________________________________________________________

Blocking Agent
Manufacturer:_____________________________________________________________
Grade: ___________________________________________________________________

Secondary Reagent
Manufacturer:_____________________________________________________________
Description (enzyme): ______________________________________________________

Substrate
Manufacturer:_____________________________________________________________
Description: ______________________________________________________________
Expiration Date: ___________________________________________________________
Wavelength Used: _________________________________________________________

Wash Buffer
Type: ____________________________________________________________________
Number of washes: ________________________________________________________
Date buffer was made:______________________________________________________
Method of washing:
❑ wash bottle
❑ multi-channel pipette
❑ multi-channel manifold
❑ automated plate washer

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14
Reconstitution of Reagents

Capture Antibody Detection Antibody Standard

Catalog Number

Lot Number

Reconstitution
Volume

Reconstitution
Buffer

Reconstitution
Date

Storage

Working Concentrations
Capture Antibody Detection Antibody Standard Secondary
Reagent

Diluting Buffer

Working
Concentration

Time Prior
to Addition

Incubation
Time

Incubation
Temperature

Sample Type:
________________________________________________________________________

Were clean pipette tips and buffer reservoirs used at each step of the assay?
❑ Yes
❑ No

Did other assays performed on the same day, using the same secondary/substrate
system, work?
❑ Yes
❑ No

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 15
Was the procedure followed according to the product insert?
❑ Yes
❑ No

If not, what was done differently?

________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

Summary of problem:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

Please attach a copy of your labeled, raw (non-zero subtracted) O.D. printout.

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16

Glossary
Analyte - the molecule being measured
Antibody - immunoglobulin with specific affinity for a particular antigen
Background - the amount of signal obtained using all reagents except the analyte
Blocking - the use of a reagent to bind non-specific sites on an ELISA plate
BSA - Bovine serum albumin, a commonly used carrier protein
Capture antibody - a primary antibody coated onto the microplate
Cell culture supernates - cell culture medium containing substances produced by cells
Curve height - the optical density (O.D.) of the highest point on the standard curve
Detection antibody - a secondary antibody, often conjugated to biotin or HRP
Detection system - a reporter system usually employing an enzyme and substrate whose end
reaction product is detected and correlated to the concentration of analyte being measured
ELISA - Enzyme-Linked-Immuno-Sorbant-Assay, a quantitative assay which utilizes the affinity of
antibodies for their antigens and an enzyme which serves as a part of the detection method
FCS - Fetal calf serum, a commonly used reagent to mimic the matrix of serum and plasma samples
HRP - horseradish peroxidase, a commonly used enzyme to modify substrate resulting in color
development
Matrix effect - a matrix effect describes an inaccurate result due to a substance in the matrix that
prevents full recovery of analyte contained in the sample. All antigens being assayed are contained in a
complex solution known as the matrix. The matrix can be simple (PBS) or complex (serum). In general,
the more complex the matrix, the more likely a matrix effect may be encountered. Refer to The
Immunoassay Handbook, edited by David Wild, Nature Publishing Group, copyright 2001, for more
information on some types of interfering substances
Microtiter plate - for matched antibody pairs, a 96-well microplate plate with flat-bottomed wells,
designed specifically for use in ELISA
Optical density (O.D.) - the absorbance of a particular substance at a specified wavelength
Plasma - a blood component obtained by collection of blood with an anticoagulant (e.g. Heparin or
EDTA), which is centrifuged to remove red blood cells (RBC), resulting in a sample that has not had the
release of clotting factors
Plate sealer - an adhesive backed plastic sheet used to protect plates
Sensitivity - the ability of a kit to discriminate between small differences of concentrations of the
cytokine/analyte being measured
Serum - a blood component obtained by allowing the blood sample to clot and removal of that clot
Solid phase - a 96-well microplate to which a capture antibody is bound
Standard - a defined, calibrated sample of the analyte being assayed, which is used to set up a curve
of known amounts against which to measure the amount of analyte in the unknown samples
Stop solution - a solution which stops the enzymatic reaction of the detection system (e.g. sulfuric
acid stops the HRP/TMB-peroxide reaction and changes the blue color to yellow, which is then read
optimally at A450)
Substrate solution - a solution containing a substance which is cleaved by an enzyme, resulting in a
color change
TMB - tetramethylbenzidine, a dye reagent used in conjunction with peroxide resulting in a blue color
when oxidized by an enzyme

4/02

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