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ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TEST
What is ELISA?
ELISA stands for enzyme-linked immunosorbent assay. This powerful antibody-based test is
used to diagnoses diseases such as HIV/AIDS and SARS and to track pathogenic agents in
water, food, and the air, wether these emerge naturally or through acts of aggression. ELISA
is also used to identify genetically modified organisms (GMOs) and to trace food allergens
and molecular markers of pregnancy and drug use.
ELISA Antibody Test
Test for the presence of antibodies to specific disease antigens in a sample of patient serum.
This type of ELISA is used to detect and diagnose infection when the atigen is undetectable
or after the body has mounted an immune respone and antibodies are present in the blood
serum.
STEP-by-STEP DESCRIPTION of ELISA
The protocols in this kit rely on indirect antibody capture ELISA. The steps in this assay are:
Step 1: Antigen is added to the wells of the
microplatestrip and incubated to allow binding,
after which unbound antigen is washed from the
wells with detergent. The detergent also serves as a
blocking agent, binding to all unused protein
binding sites in the wells and preventing
nonspecific binding of antibody.
Step 2: Primary antibody (sample serum)
solution is added to the wells and incubated to
allow the antibody to bind to the antigen. The
unbound primary antibody is washed from the
wells.
Step 3: Enzyme-labeled secondary antibody
solution is added to the wells and incubated to
allow the secondary antibody to bind to the
primary antibody. The unbound secondary
antibody is washed from the wells.
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Step 4: Chromogenic (color-producing) enzyme
substrate is added to the well and incubated to allow
color to develop. Results of the assay are evaluated.
Wells that remain colorless are negative and wells
that turn blue are positive.
GENERAL INTRODUCTION TO THIS ELISA KIT
Microplate strips: Microplates are made of polystyrene which absorbs (binds) proteins by
hydrophobic interaction. The plates provided in this kit have 96 wells, arranged in 8
removeable rows of 12-well strips. Each well holds approximately 250 microliters (l).
Antigen: In this kit, the antigen is chicken gamma-globulin (purified from egg yolks) which
serves as a generic representative of any hypothetical antigen, protein or otherwise.
Incubation times: The rate of binding depends on the incubation temperature and the
concentrations of the reagents. This kit has been optimized so that each incubation can be
performed for 5 minutes at room temperature. Exceeding this time or temperature will cause
an increase in color intensity and possibly some background color in the negative controls.
Blocking: blocking agents are added after antigen adsoroption to prevent nonspecific binding
of antibodies to the plastic, which would produce false positive results. The blocking agent
may be a protein or a detergent (or both). Common blocking agents include Tween 20 (a
nonionic detergent that is used in this kit), nonfat dry milk, gelatin, and bovine serum
albumin (BSA). Although Tween 20 is a sufficient block for this protocol, you may wish to
add the following blocking step for teaching purposes: have the students add 50 l of 1%
gelatin in wash buffer to their wells for 15 min after the addition of the antigen and then
perform a wash step.
Primary (1) antibodies: The antibodies that recognize and bind to the antigen in an
immunoassay are primary antibodies.
Sencondary (2) antibodies: Secondary antibodies recognize and bind to primary antibodies.
They are made in animals of a different species than that used to make the primary antibody.
Colorimetric detection: Secondary antibodies for ELISA are linked to enzymes. Detection
of secondary antibodies that are bound to primary antibodies occurs by an enzyme-substrate
reaction. In this kit, the 2 antibody is linked to horseradish peroxidase (HRP). In the
presense of hydrogen peroxide (H2O2), HRP catalyzes the oxidation of the chromogenic
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substrate 3,3’,5,5’-tetramethylbenzidene (TMB). This oxidation of TMB by HRP forms a
blue product.
Note: TMB is light sensitive, and the assay results should be determined 5-10 minutes after
the substrate is added to the wells. If the microplate strips sit longer, nonspecific color may
develop. Color that develops after the 5-minute incubation should not be sonsidered in the
assay results. After 20-30 minutes, the blue color may begin to fade as TMB precipitates out
of solution.
Controls: Controls are always run side by side with actual samples to make sure that the
procedure is working correctly. Controls can resolve ambiguous results that occur due to
human error or contaminated reagents; controls must be included in any valid ELISA. For
the negative control, the antigen or primary antibody is either omitted (as in this kit) or the
antigen is replaced by a factor that will not bind specifically to the antibody. The positive
control always contains the target antigen or antibody
Analysis of Results:
Qualitative results can be determined visually without the use of complicated
instrumentation.
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Quantitative results can be estimated visually and scored symbolically, e.g. (++): strong
signal, (+): weak signal, (+/-): an ambiguous signal, and (-): no detectable signal. For
accurate and precise detemination of concentrations, a microplate reader is required.
Microplate readers quantitate the absorbance of light by the colored substrate in each well
of a microplate. They use the negative control wells to set a baseline and then read the
absorbance of each well at a specified wavelength. For example, the peak absorbance for
TMB is at 655 nm.
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LABORATORY PREPARATION
Objectives
Step 1. Prepare buffers
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary antibody to
make 50 stocks
Step 3. Dilute 50 stock solutions
Supplied Reagents Quantity
Antigen, chicken gamma globulin, freeze-dried 1 vial
Primary antibody/serum sample, rabbit anti-chicken polyclonal
antibody, freeze-dried 1 vial
Secondary antibody, goat anti-rabbit antibody conjugated to HRP,
freeze-dried 1 vial
HRP enzyme substrate, TMB 1 bottle
10 phosphate buffered saline (PBS) 1 bottle
10% Tween 20 1 bottle
Required Reagents
Distilled water, sterile is recommended 1L
Step 1. Prepare buffers.
We recommend you use a 100 ml and a 1 L graduated cylinder for preparing the buffer
solutions. You will also need 1 L of distilled water.
Buffer Volume Reagent Used for
1 PBS, 90 ml Distilled water Rehydrating antigen,
primary and secondary
100 ml 10 ml 10 PBS
antibodies to make 50
reagent stock solutions
Diluting 50 antigen
Wash buffer, 805 ml Distilled water Diluting 50 primary
antibody stock for positive
900 ml 90 ml 10 PBS
control and positive student
4.5 ml 10% Tween 20 samples
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Negative controls
Negative student serum
samples
Dilution of 50 antibody
stocks
Plate washing
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary antibody.
Carefully remove the stoppers form the three freeze-dried reagents and use a distilled glassed
pipet (DGP) to add 0.5 ml 1 PBS to each. Close the stoppers and shake to mix. These
solutions are 50 concentrates, or stock solutions. NOTE: You must NOT use wash buffer
in this step.
Freeze-dried Protocol for 50 Stock Used for
Reagent Solution
Antigen Add 0.5 ml of 1 PBS to vial Purified antigen
Primary antibody Add 0.5 ml of 1 PBS to vial Positive controls
Positive student serum
samples
Secondary antibody Add 0.5 ml of 1 PBS to vial Secondary antibody
Step 3. Dilute 50 stock reagents.
Label one 50 ml bottle or tube for each of the diluted solutions below. Use a DGP to add the
contents of the appropriate 50 concentrated stock to the corresponding 50 ml bottle or tube.
Diluted Solution Volume Reagent Used for
1 antigen, label one 50ml 24.5 ml 1 PBS Purified antigen
bottle or tube
0.5 ml 50 antigen
NOTE: you must NOT add any buffer containing
Tween 20 to the antigen, or the experiment will not
work.
Use the DGP to rinse out the vial with some of the
diluted reagent to ensure that all of the stock
solution is used.
Close the cap and shake to mix.
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1 serum (1 primary 24.5 ml Wash buffer Positive controls
antibody), label one 50 ml
0.5 ml 50 primary Positive student
bottle or tube
antibody stock serum samples
Use the DGP to rinse out the vial with some of the
diluted reagent to ensure that all of the stock
solution is used.
Close the cap and shake to mix.
1 secondary antibody, label 24.5 ml Wash buffer Secondary antibody
one 50 ml bottle or tube
0.5 50 secondary Antibody stock
antibody stock
Dilute the secondary antibody less than 24 hours
before the start of the lesson.
Use the DGP to rinse out the vial with some of the
diluted reagent to ensure that all of the stock
solution is used.
Close the cap and shake to mix.
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LABORATORY PROCEDURE
Step 1: Using a pipet, 50 l of purified disease antigen is added to the wells of the microplate
strip and incubated for 5 minutes, allowing antigen to bind to the wells. The wells are rinsed
with wash buffer (PBST: phosphate buffered saline containing 0.0% Tween 20) to block the
unoccupied protein binding sites in the wells.
Step 2: Serum samples and positvie and negative controls (50 l) are added to the wells and
incubated for 5 minutes at room temperature. If antibodies against the disease are present in
the serum sample, they will bind to the purified disease antigen already bound in the wells.
The wells are rinsed with wash buffer to remove unbound antibody.
Step 3: Horseradish peroxidase (HRP)-labeled secondary antibody (50 l) is added to the
wells and incubated for 5 minutes at room temperature. The secondary antibody is antibody
that recognizes and bind to the primary antibody. Wells are rinsed with wash buffer to
remove unbound secondary antibody.
Step 4: The enzyme substrate (50 l) is added to each well and students watch color
development. If HRP is present (meaning that antibodies to the purified antigen were present
in the serum sample), the solution in the wells will turn blue within 5 minutes, indicating a
positive diagnosis. If there were no antibodies to the disease antigen, the wells will remain
colorless, indicating a negatvie diagnosis.
1. Label 3 wells with a “+” for the
positive controls and 3 wells with
a “” for the negative controls.
2. Use a fresh pipet tip to transfer 50 l of purified
antigen (Ag) into all wells of the micorplate.
3. Wait 5 minutes for the antigen to bind to the
plastic wells.
4. Wash:
a. Tip the microplate strip upside down
onto the paper towels, and gently tap the
strip a few times upside down. Make
sure to avoid splashing sample back into
wells.
b. Discard the top paper towel.
c. Use your transfer pipet to fill each well
with wash buffer, taking care not to spill
over into neighboring wells. Note: the
same transfer pipet is used for all
washing steps.
d. Tip the microplate strip upside down onto the paper towels and tap.
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e. Discard the top 23 paper towels.
5. Repeat wash step 4.
6. Use a fresh pipet tip to transfer 50 l of the positive
control (+) into the three “+” wells.
7. Use a fresh pipet tip to transfer 50 l of the negative
control () into the three “” wells.
8. Transfer 50 l of serum samples into the other wells,
using a fresh pipet tip for each serum sample.
9. Wait 5 minutes for the antibodies to bind to their
targets.
10. Wash the unbound primary antibody out of the wells
by repeating all of wash step 4 two times.
11. Use a fresh pipet tip to transfers 50 l of secondary antibody into all wells of the
microplate strip.
12. Wait 5 minutes for the antibodies to bind to their targets.
13. Wash the unbound secondary antibody out of the wells by repeating wash step 4 three
times.
14. Use a fresh pipet tip to transfer 50 l of enzyme substrate into all wells of the
microplate strip.
15. Wait 5 minutes. Observe and record the results.
