ELISA Disease Detection

Modeling Antigen and Antibody Detection

3/30/2020 Ver. A
Enzyme-Linked ImmunoSorbant Assay (ELISA)
ELISAs use antibodies to detect proteins in a patient’s
sample.

Many diseases and conditions can be detected and

diagnosed with ELISAs, including:
• Influenza
• HIV
• SARS
• Zika virus
• West Nile virus
• Lyme Disease

Researchers are also developing ELISAs that can be

used to diagnose current or past coronavirus
(SARS-CoV-2) infection. Novel Coronavirus SARS-CoV-2
This scanning electron microscope image shows SARS-CoV-2
(round gold objects) emerging from the surface of cells cultured
in the lab. SARS-CoV-2, also known as 2019-nCoV, is the virus
that causes COVID-19. The virus shown was isolated from a
patient in the U.S. Credit: NIAID-RML

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Antigen-Antibody Interactions
Antigen
Pollen, bacteria, viruses, and other foreign molecules are
seen by your body as invaders, and they create an
immune response. These foreign invaders are called Variable
antigens. Region

Your immune system makes antibodies in response to

antigens. The antibodies bind antigens, flagging them for
destruction by immune cells. Constant
Region
Antibodies have two regions: variable and constant. Each
tip of the “Y” in the variable region is highly specific and
binds to only one particular antigen. The constant region is Antibody
the same for every antibody of the same type (there are 5
different types of antibodies).

Antibodies can also be produced by injecting an animal

with antigen - disease agents or even antibodies from a
different type of animal.

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Infection Timeline — Antigens and Antibodies
After infecting a person, a virus
multiplies in the body and the
concentration of viral antigens
increases.

Soon after infection, the immune

system kicks in and starts producing
antibodies.

As the antibody concentration

increases, antibodies bind to viruses
and target them for destruction by
the immune system.

Eventually, the viral antigen

concentration drops, the infection is
cleared, and the person recovers.

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Antigen Detection ELISA Components
● Antigen — in an antigen detection ELISA, the Antigen
patient sample is tested for the presence of (virus)
antigens from viruses, bacteria, etc.
● Primary antibody — binds to the antigen
○ can be produced in a lab by injecting the target Primary
antigen into an animal and then harvesting the Antibody
serum (rabbit anti-virus)
● Secondary antibody — binds to the constant
region of the primary antibody
○ made by injecting the primary antibodies from
Secondary
one animal into a different animal
Antibody
○ secondary antibodies are attached to an (goat anti-rabbit)
enzyme which catalyzes a color change when
substrate is added
Enzyme
● Substrate — changes color in the presence of the
enzyme, indicating a positive result Substrate

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Influenza Antigen Detection
Influenza binds to target cells via the Antigen
hemagglutinin glycoprotein, HA. Scientists can (influenza HA)
create antibodies that detect and bind to
influenza by injecting HA into an animal.

Primary
Antibody
(rabbit anti-influenza
HA)

Secondary
Antibody
(goat anti-rabbit)

cross-section of influenza virus Hemagglutinin Enzyme

https://www.cdc.gov/flu/resource-center/freereso
urces/graphics/images.htm (HA)
Substrate

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Antigen Detection ELISA Animation
Antigen Detection ELISA Animation
(click on link to view animation, or use presentation mode to play the embedded video)

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Model an Antigen Detection ELISA
On the next slide:

Model the process of an ELISA showing the final result.

Use the following slides to assist you and guide you through
the steps.

Make sure to use all components provided (shown to the right),

your model should show:
Primary Secondary
Antigens
Antibody Antibody
Primary Antibody

Enzyme-Linked Secondary Antibody

Substrate
Antigens Substrate

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Digital ELISA Model

ELISA well
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Antigen Detection ELISA – Step 1
Assay Step 1 — Bind antigen to the well
a. Add the patient sample to the well. This sample contains many
different proteins that may or may not contain the antigens that you
are trying to detect. These proteins bind non-specifically (adsorb) to
the plastic well, due to hydrophobic interactions.
b. Incubate the sample for 5 minutes, then tap out the fluid.
c. Add wash buffer to the well to rinse out anything that isn’t bound, and
to block the inner surface of the well. This prevents anything from
binding non-specifically in future steps.
d. Repeat the wash step.
Model Step 1
a. Add the antigens around the bottom edge of your blank paper “well”.
These represent the proteins / antigens that might be present in any
given patient sample.

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Antigen Detection ELISA – Step 2
Assay Step 2 — Bind primary antibody to antigen
a. Add the primary antibodies to the well. The antibodies bind only to a
specific antigen out of the many that may be bound to the well. For
example, an anti-HIV antibody would only bind to HIV antigen. This
antigen-antibody interaction is specific and strong.
b. Incubate the sample for 5 minutes, then tap out the fluid.
c. Add wash buffer to the well to rinse out anything that isn’t bound. If
the primary antibody did not bind to any antigen, then it will be
washed away in this step.
d. Repeat the wash step.
Model Step 2
a. Add the primary antibodies into your well. Overlay the antibody so
that the black regions align. This represents specific antibody-antigen
binding.
b. Model the wash step by removing the unbound primary antibody.

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Antigen Detection ELISA – Step 3
Assay Step 3 — Bind secondary antibody
a. Add the enzyme-linked secondary antibodies to the well. These
antibodies bind tightly to any primary antibodies that are present. The
secondary antibodies are covalently linked to an enzyme, horseradish
peroxidase, which will catalyze a reaction with a substrate to produce
a color change.
b. Incubate the sample for 5 minutes, then tap out the fluid.
c. Add wash buffer to the well to rinse out any secondary antibodies that
did not bind.
d. Repeat the wash step 2 more times (3 times total).
Model Step 3
a. Add the secondary antibodies into your well. Overlay the secondary
antibody so that the patterns align with the primary antibody.
b. Model the wash step by removing the unbound secondary antibody.

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Antigen Detection ELISA – Step 4
Assay Step 4 — Add enzyme substrate for detection

a. Add the substrate to the well, wait 5 minutes, and evaluate results. If
the antigen was present, then the primary and secondary antibodies
bound and the well will turn blue. If there was no antigen, then no
primary or secondary antibodies bound, so the well will remain clear.

Model Step 4

a. Add the substrate into your well. Align the substrate with the enzyme
on the secondary antibody.

b. At this point, the enzyme catalyzes a reaction where the substrate

turns blue - a positive result.

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Questions — Antigen Detection ELISA
1. Examine this graph of antigen and antibody concentrations over
time.
a) Could you use an antigen detection ELISA to accurately
diagnose a patient on day 35? Explain your answer.
• No, you cannot use an antigen detection ELISA on
day 35 because antigen levels are too low to detect
at that stage of infection.

a) Antigen detection ELISAs detect viral proteins. Viral DNA

or RNA can be detected using a different technique called
PCR (polymerase chain reaction). Using the graph,
explain when a viral PCR test would be most useful.
• A viral PCR test would be useful early in the
infection, when viral replication is high and viral DNA
or RNA is abundant. Based on the graph, PCR
would be most effective between day 0 and day 15,
when antigen levels are high, indicating active viral
presence.
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Questions — ELISA Design
2. Refer to the diagram, and notice where the primary
antibody binds antigen and secondary antibodies.
Antigen
(Influenza HA)
a) Could goat anti-rabbit secondary antibodies be
used to detect two different types of rabbit
antibodies? Explain your answer.

• Yes, goat anti-rabbit secondary antibodies Primary

can detect different rabbit antibodies Antibody
because they can bind the constant region (rabbit anti-influenza
common to all rabbit antibodies. HA)

a) If HIV antigen was injected into the rabbit instead

of influenza, could the same secondary Secondary
antibodies (goat anti-rabbit) be used to detect Antibody
both rabbit anti-influenza antibodies AND rabbit (goat anti-rabbit)
anti-HIV antibodies? Explain your answer.

• Yes, the same secondary antibodies can Enzyme

detect both anti-influenza and anti-HIV
rabbit antibodies since they target the Substrate
constant region which is the same ford
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4.
Question — ELISA Design
You modeled an indirect ELISA, which uses enzyme-linked secondary antibodies. In a
direct ELISA, the enzyme is linked to the primary antibody instead, so no secondary
antibody is required.

Look at your model and think about what might happen if the enzyme was attached to
the primary antibody directly, so that a secondary antibody was not needed.

a) Would you still get a blue color change when the substrate was added? Explain your
answer.

• Yes, you would still get a blue color change when the substrate is added because the
enzyme is still present and catalyze the reaction.

b) How might the signal intensity (amount of color change) compare to the indirect
ELISA that you modeled first?

• The signal intensity might be lower compared to an indirect ELISA because each
primary antibody in the direct ELISA has only one enzyme attached, whereas in the
indirect method, multiple secondary antibodies can bind to each primary antibody,
amplifying the signal.

c) What might be one advantage to using an enzyme-linked secondary antibody?

• An advantage of using an enzyme-linked secondary antibody is signal amplification, as

16 multiple secondary antibodies can bind to one primary antibody, increasing the color
intensity.
Antibody Detection ELISA Overview
ELISAs can also use purified antigen to detect
antibodies in a patient’s serum. The presence of viral
antibodies in a patient sample indicates a current or
previous infection.

Coronavirus (SARS-CoV-2) binds to target host cells

via its spike glycoprotein, S. When someone is infected
with coronavirus, their immune system makes
antibodies to viral proteins, including S protein.

Scientists are developing ELISAs that use purified S

protein as the antigen to detect the presence of Spike
coronavirus antibodies in a patient’s serum. ELISAs Glycoprotein
that detect other viral proteins are being researched as
(S)
well.

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Coronavirus Antibody Detection ELISA
Coronavirus Antibody Detection ELISA
Antigen
(lab-purified coronavirus S
Coronavirus Infection protein)

Coronavirus Patient Sample

infects person (anti-coronavirus antibodies
will be present in sample if
patient was infected)
Person makes
antibodies against Secondary Antibody,
coronavirus goat anti-human
(binds if anti-coronavirus human
antibodies are present in the sample)

Enzyme
Substrate

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Antibody Detection ELISA Animation
Antibody Detection ELISA Animation
(click on link to view animation, or use presentation mode to play the embedded video)

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Model an Antibody Detection ELISA
On the next slide:

Model the process of an ELISA showing the final result.

Use the video from the previous slide to guide you through the
steps.

Make sure to use all components provided (shown to the right),

your model should show:
Primary Secondary
Antigens
Antibody Antibody
Primary Antibody

Enzyme-Linked Secondary Antibody

Substrate
Antigens Substrate

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Digital ELISA Model

ELISA well
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Questions — Antibody Detection ELISA
After antigen is added, the wells are washed. Why is the wash step important? Give two reasons.

1. It removes ubound antigen, preventing false positives.

2. It ensures that only specific binding interactions are detected in later steps.

Next, the patient sample is added. Patient samples contain a variety of antibodies. How is the Coronavirus
detected?

• The Coronavirus is detected if specific antibodies in the patient sample bind to the viral antigen
coated on the well.

An enzyme-linked secondary antibody is added next followed by the addition of substrate. If the sample
turns blue (a positive result) the patient sample contains antibodies to coronavirus. Does this necessarily
indicate an ongoing infection? Explain your answer.

• No, a positive result indicates the presence of antibodies, which could be from a past infection, not
necessarily an ongoing one.

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Questions — Antibody Detection ELISA
According to this graph, could you use an antibody
detection ELISA to accurately diagnose a patient 35 days
after exposure? Explain your answer.
• No, you cannot use an antigen detection ELISA on day
35 because antigen levels are too low to detect at that
stage of infection.

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