Enzyme-linked immunosorbent assay

(ELISA)
BMLT 457

Gloria Agyekum Boaitey (Ph.D.)

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 Introduction
• ELISA is the acronym for Enzyme-Linked Immunosorbent Assay
• ELISA is also known as an enzyme immunoassay (EIA)
• ELISA is a very sensitive method used to detect the presence of
antigens or antibodies of interest in a biological sample
• ELISA has been used successfully for the determination of limitless
number of important compounds in biological samples
• ELISA can detect the presence and quantity of proteins, such as
hormones and antibodies and bacteria or viruses
• In this immunodiagnostic technique, the label used is an enzyme

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 Introduction
• The ELISA assay uses the coupling of antigens and antibodies
• It relies on the specificity and affinity of antibodies for antigens
• Specificity is the ability to discriminate among diverse proteins
• Affinity is the ability to tightly bind to molecules
• To determine how much antibody is present in a sample, an antigen is
incorporated into the reagent

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 History of ELISA
• In 1971, Peter Permann and Eva Engvall in Stockholm published the
first paper on ELISA showing they could quantify the amount of IgG in
rabbit serum using alkaline phosphatase as the reporter label
• The same year, Anton Schuurs and Bauke Van Weemen in the
Netherlands published a paper showing that with an Enzyme
Immunoassay (EIA), they could quantify the amount of human
chorionic gonadotropin in urine with horseradish peroxidase coupled
with glutaraldehyde as the reporter label
• The assays were highly sensitive and compared favorably with
Radioimmunoassay (RIA)

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 History of ELISA

• In 1976, Competitive ELISA technique in which a conjugated substrate

competes with a protein of interest for binding sites was used to
detect human chorionic gonadotropin hormone
• In 1978, Indirect ELISA was developed and used to detect human
serum bilirubin
• ELISA was the first screening test which was commonly used for HIV
testing. ELISA was approved for HIV testing on March 2, 1985
• Currently, ELISA is used to detect antibodies of SARS-CoV-2

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Separation matrix (96 well
microtiter plate)

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Microplate reader

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 Types of ELISA methods

• There are five types of ELISA methods which include:

• Direct ELISA
• Indirect ELISA
• Sandwich ELISA
• Competitive ELISA
• Multiplex ELISA
• The indirect and sandwich ELISA methods are the most common

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 Examples of enzymes used in
ELISA
• Alkaline phosphatase
• Horseradish peroxidase
• β-Galactosidase
• Catalase
• Acetylcholinesterase

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 Procedure of Indirect ELISA
• Binding Known Antigen - The indirect ELISA method begins with a
sample of known antigen solution (coating reagent) being bound to
the wells of a microtiter plate
• Incubation- The plate is covered with an adhesive and incubated for 2
hours at room temperature or 4°C overnight
• Blocking - The other unoccupied sites in each well are then blocked
by a blocking buffer (concentrated solution of non-interacting
protein, like casein or bovine serum albumin), to prevent other
proteins in the test sample from adhering
• Incubation- The plate is covered with an adhesive and incubated for 2
hours at room temperature
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 Procedure of Indirect ELISA
• Washing – Rinse with PBS to remove any unbound antigen
and non-interacting protein
• Adding Test Sample Primary Antibody (Coating) - The test
sample of serum containing the primary antibodies is added
to each well. Antibodies could be HIV, measles, or hepatitis B
antibodies, for example
• Incubation- The plate is covered with an adhesive and
incubated for 2 hours at room temperature
• Washing – Rinse to remove any antibodies that did not bind
to the known antigen

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 Procedure of Indirect ELISA
• Adding Enzyme-linked Secondary Antibody - An enzyme-
linked secondary antibody is added next to bind to the test
sample antibodies. The enzyme on the secondary antibodies
are proteins, such as horse radish peroxidase or alkaline
phosphatase
• Incubation- The plate is covered with an adhesive and
incubated for 2 hours at room temperature
• Washing – Rinse to remove any secondary antibodies that
did not bind to the primary antibody
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 Procedure of Indirect ELISA
• Adding Substrate - A substrate is then applied which is
converted by the enzyme to give a color or fluorescence or
electrochemical signal
• In the presence of horse radish peroxidase, ABTS (2,2'-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) turns green, OPD
(o-phenylenediamine dihydrochloride) turns orange, and
TMB (3,3',5,5'-tetramethylbenzidine) turns blue
• In the presence of alkaline phosphatase, pNPP (p-nitrophenyl
phosphate) substrate turns yellow

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 Procedure of Indirect ELISA
• Incubation – Incubate at room temperature for 1 hour
• Reading Results - By using a spectrophotometer,
spectrofluorometer, or electrochemical device, the results
can be read and recorded
• The amount of color produced is proportional to the amount
of primary antibody bound to the antigen proteins on the
bottom of the wells

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Procedure of Indirect ELISA

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 ELISA data interpretation
• ELISA can generate 3 types of data:
• Qualitative – ELISA only tells whether a target is present or
absent and is reported as positive or negative. Here, ELISA
compares absorbances obtained in the procedure with a
cutoff value
• Quantitative & Semi-Quantitative – ELISA data can be
interpreted by comparing absorbances obtained with a
standard curve (serial dilution of known, purified antigen
dilutions with concentrations on the x-axis vs absorbances on
the y-axis) in order to calculate the concentration of the
unknown antigen in the sample
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 Advantages of Indirect ELISA
• High sensitivity
• High specificity
• Different primary detection antibodies can be used with a
single labelled secondary antibody
• It is cost effective because fewer labelled secondary
antibodies are needed

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Disadvantages of Indirect ELISA
• Cross-reactivity can occur with the secondary antibody
leading to non-specific signals
• It requires many incubation steps

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 Assignment
• Watch tutorial overview of Direct, Indirect, and Sandwich ELISA, go to
https://www.youtube.com

• Watch animation of Indirect ELISA method and test results, go to

https://www.youtube.com
• Read on the ELISpot procedure

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 Procedure of Direct ELISA
• This is the simplest type of ELISA where a solution containing
antigens to be tested are bound to microtiter wells
• The unoccupied sites in each well are then blocked using a
protein solution (blocking buffer) to prevent other proteins in
the test sample from adhering to the walls of the plate
• An enzyme is conjugated to an antibody in a separate
reaction and the enzyme-antibody complex is applied to bind
to the antigen
• Washing is carried out to remove excess enzyme-antibody
complex
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 Procedure of Direct ELISA
• Antigen-enzyme-antibody complex is incubated
• Substrate is added which is converted by the enzyme to a
detectable coloured product
• The coloured product is measured using a
spectrophotometer or other appropriate instruments to give
a quantitative value to the coloured product

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 Advantages of Direct ELISA
• This technique is faster than other ELISA techniques as fewer
steps are required
• It is less prone to error since fewer reagents and steps are
needed, i.e. no potentially cross-reacting secondary antibody
needed

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 Disdvantages of Direct ELISA
• It is less sensitive compared to the Indirect and Sandwich
methods
• It makes measurement of crude samples difficult, since
contaminating proteins can compete for plastic binding sites

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 Sandwich ELISA
• Many commercially prepared ELISA are based on the
Sandwich method
• Sandwich ELISA quantifies antigens between two layers
of antibodies
• The antigen to be measured must have at least two
antigenic epitopes capable of binding to the antibody
because at least 2 antibodies act in the Sandwich
method

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 Sandwich ELISA
• Either monoclonal or polyclonal antibodies can be used as
the capture and detection antibodies in this method
• Monoclonal antibodies recognize a single epitope, and this
allows for quantification of small differences in antigens.
They are often used as detection antibodies
• Polyclonal antibodies are often used as capture antibodies
because they can pull down more antigens

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 Procedure of Sandwich ELISA

(1) Plate is coated with a capture antibody; (2) sample is added, and any antigen
present binds to capture antibody; (3) detecting antibody is added and binds to antigen;
(4) enzyme-linked secondary antibody is added and binds to detecting antibody; (5)
substrate is added and is converted by enzyme to detectable form.
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 Procedure of Sandwich ELISA
• The Sandwich ELISA method begins with a sample of known antibody
solution being captured on the wells of a microtiter plate
• The other unoccupied sites in each well are then blocked by a
blocking buffer to prevent other proteins in the test sample from
adhering
• A sample containing antigen is added and allowed to react with the
immobilized antibody
• Washing to remove any unbound antigen and non-interacting protein
• Detecting antibody is added and allowed to bind to the antigen

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 Procedure of Sandwich ELISA
• Enzyme-linked secondary antibody is added and allowed to
bind to the detecting antibody
• Washing to remove unbound antibody-enzyme conjugate
• Substrate is added which is converted by the enzyme to a
detectable coloured product
• Absorbance signal is measured to detect the presence and
quantity of antigen in the sample wells

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 Applications of Sandwich ELISA
• Cancer screening
• Drug test
• Pregnancy test
• Detection of food allergens
• Virus detection (e.g: HIV)

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 Advantages of Sandwich ELISA
• High sensitivity
• High specificity
• Reagents are cheap and have long shelf life
• Easy and quick to perform
• Suitable for complex samples as antigen does not require purification
prior to measurement
• It has good flexibility because it uses both direct and indirect ELISA
methods

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 Disadvantage of Sandwich
ELISA
• Antibody optimization can be difficult. Only products specifically
tested for Sandwich ELISA can be used since antibody must
specifically bind to an epitope of the antigen and not cross-react with
its partners

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 Competitive ELISA
• Competitive ELISA is also known as Inhibition ELISA
• It measures an antigen’s concentration through the detection
of signal interference
• Some Competitive ELISA kits use labelled antigens instead of
labelled antibodies. This orientation is called “antibody
down”

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Procedure of Competitive ELISA
• A sample of known antibody solution is captured on the
wells of a microtiter plate
• The other unoccupied sites in each well are then blocked by
a blocking buffer to prevent other proteins in the test sample
from adhering
• A sample containing an unknown quantity of unlabeled
antigen is added to the sample wells coated with antibody
• Next, an enzyme-linked (labeled) control antigen is also
added to the sample wells

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Procedure of Competitive ELISA
• If the sample antigen is more, less of the labelled control
antigen will bind to the antibody so generate less coloured
product
• If the sample antigen is low, more of the labelled control
antigen will bind to the antibody and generate more
coloured product
• Because the standard curve in a competitive ELISA exhibits
the maximum signal at the lowest concentrations of sample
antigen, this assay is very sensitive

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Procedure of Competitive ELISA

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 Advantages of Competitive
ELISA
• Impure samples can be used as no sample processing is
needed
• High specificity
• Flexible procedure: it can be based on direct, indirect or
sandwich procedure
• Very robust: minimizes the effects of sample dilution and
sample matrix effects

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Disdvantages of Competitive
ELISA
• Very complex protocol which is time consuming
• Limited conjugated antibodies

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