DEMONSTRATION OF ENZYME

LINKED IMMUNOSORBENT
IMMUNOSORBENTASSAY (ELISA)
Introduction
• Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA) is a
sensitive immunochemical technique for the detection and quantification of
hormones, peptides, proteins and antigens.

• It is widely utilized in medicine as diagnostics and as a measure of quality control

in industries and research.
• The assay is performed on a solid matrix using enzymes that are linked to an
antibody which would serve as a marker for the detection. Therefore, ELISA is
also known as solid-phase enzyme immunoassay.

• An enzyme conjugated with an antibody reacts with a colorless substrate to

generate a colored reaction product. Such a substrate is called a chromogenic
substrate.

• A number of enzymes have been employed for ELISA, including alkaline

phosphatase, horseradish peroxidase, and β-galactosidase.
Objectives
• After the completion of this exercise you will be able to:
- explain the principle and immuno-quantification technique of ELISA, and
- detect and quantify peptides, proteins, antibodies and hormones.
Materials Required

• ELISA plate,
• Antigen,
• standard polyclonal anti-serum/antigen-specific monoclonal antibody,
• Low and high titer serum specific to the antigen,
• Substrate solution,
• Coating buffer (pH 9.6),
• Washing buffer (pH 7.4),
• Stop solution (1M H2SO4),
• 1.5 % Skimmed milk powder solution and/0.5% BSA (Bovine Serum Albumin).
Preparation of Solutions
• Coating buffer (pH 9.6): Weigh 0.29 g Sodium bicarbonate + 0.15 g Sodium
chloride + 0.02 g Sodium azide and dissolve in distilled water, making up the
volume to 1L.

• Substrate buffer: Weigh 1.45 g disodium hydrogen phosphate + 8.0 g Sodium

chloride + 0.20 g Potassium chloride + 0.20 g Potassium dihydrogen phosphate.
Dissolve in 1L distilled water and add 500 µl of TWEEN-20.

• Phosphate Buffered Saline (PBS) – pH 7.2, 0.15 M: Dissolve 8 g Sodium chloride +

0.2 g Potassium chloride + 1.15 g Disodium hydrogen phosphate + 0.2 g
Potassium dihydrogen phosphate in 500 ml Distilled Water. Adjust the pH to 7.2
and make up the volume to 1000 ml with distilled water.
• Citrate Buffer – pH 5.0, 0.1M: 33 ml of 0.1 M Citric acid + 67 ml of 0.1M Sodium
citrate.

• Tris-HCl Buffer – pH 7.6, 0.01 M: Dissolve 1.21 g Tris in 50 ml distilled water and
adjust the pH to 7.6 with HCl. Make up the final volume to 100 ml. Dilute 10
times before Use.

• ELISA Substrate:
1. Diaminobenzidine (DAB): Dissolve 6 mg Diaminobenzidine in 10 ml 0.1M Tris-HCl
buffer (pH 7.6). Add 10 µl of Hydrogen peroxide [just before use].
2. Ortho-Phenyl-diamine (OPD): Dissolve 34 mg Ortho-phenyl-diamine in 100 ml of
0.1M Sodium citrate Buffer (pH 5.0). Add 50 µl of Hydrogen peroxide [just before
use].
Principle
• Enzyme-Linked Immunosorbent Assay (ELISA) follows the basic principle of
antibody binding to a specific epitope of antigens.

• The assay involves three principle reactions-

First, specific immune reaction (Antigen-antibody reaction)
Second, enzymatic chemical reaction of converting substrate (chromogen)
to an insoluble coloured product.
Third, signal detection of colour and quantification of colour intensity.
• There are four major types of ELISA:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA

• These allow qualitative detection or quantitative measurement of either antigen

or antibody.

• Each type of ELISA can be used qualitatively to detect the presence of antibody
or antigen.

• Alternatively, a standard curve based on known concentrations of antibody or

antigen is prepared, from which the quantitative measurement of unknown
concentration of a sample either antigen or antibody can be determined.
Direct ELISA
• Direct ELISA is the simplest form of ELISA.

• In direct ELISA, primary antibody labelled with a conjugated enzyme directly

binds to an antigen.

• The addition of chromogenic substrate produces colour change on enzyme

hydrolysis.
 Direct ELISA

Figure depicting the direct ELISA. For the detection of antigen in a given biological sample, enzyme (e.g. Horse reddish
peroxidase) conjugated antibody is used. When antigen is detected, chromogenic substrate produces colour. The
intensity of the colour can be measured by using a machine called ELISA plate reader at particular wavelength.
Indirect ELISA
• In this technique, the antigen to be detected and quantified is immobilized on to
a plate using a coating antibody that specifically traps the antigen on the solid
phase.

• A secondary antibody, labelled with an enzyme (termed as reporter enzyme) is

then directly allowed to bind with the immobilized antigen.

• The presence of antigen in test sample is then detected by introducing an

enzyme- substrate (chromogen) to the antigen-antibody-enzyme complex, which
produces the colour.
Indirect ELISA

Antigen is detected by two step process. First, primary antibody specific for antigen to be detected, are
added and then secondary antibody conjugated with enzyme is allowed to bind with primary antibody.
Enzyme (e.g. Horse reddish peroxidase) works on its substrate and colour is produced for which intensity can
be measured spectrophotometrically (Using ELISA plate reader at particular wavelength).
INDIRECT ELISA
• Antibody can be detected or quantitatively determined with an indirect ELISA.

• Serum or some other sample containing primary antibody (Ab1) is added to an

antigen-coated microtiter well and allowed to react with the antigen attached to
the well. After any free Ab1 is washed away, the presence of antibody bound to
the antigen is detected by adding an enzyme-conjugated secondary anti-isotype
antibody (Ab2), which binds to the primary antibody.
• Any free Ab2 then is washed away, and a substrate for the enzyme is added.

• The amount of colored reaction product that forms is measured by specialized

spectrophotometric plate readers, which can measure the absorbance of all of
the wells of a 96-well plate in seconds.
• Indirect ELISA is the method of choice to detect the presence of serum antibodies
against human immunodeficiency virus (HIV), the causative agent of AIDS.

• In this assay, recombinant envelope and core proteins of HIV are adsorbed as
solid-phase antigens to microtiter wells. Individuals infected with HIV will
produce serum antibodies to epitopes on these viral proteins.

• Generally, serum antibodies to HIV can be detected by indirect ELISA within 6

weeks of infection.
Variations in the enzyme-linked immunosorbent assay (ELISA) technique allow determination of antibody or antigen. Each
assay can be used qualitatively, or quantitatively by comparison with standard curves prepared with known concentrations
of antibody or antigen. Antibody can be determined with an indirect ELISA (a), whereas antigen can be determined with a
sandwich ELISA (b) or competitive ELISA (c). In the competitive ELISA, which is an inhibition-type assay, the concentration
of antigen is inversely proportional to the color produced.
Sandwich ELISA
• In sandwich ELISA, the antigen is sandwiched between 1o and 2o antibody. This
assay can be done in a direct way or indirect way.

• The primary antibody (1o) is immobilized on the plate to absorb the antigen of
interest.

• The secondary antibody, which is enzyme-conjugated is then introduced to bind

with the antigen trapped on the 1° antibody.

• The addition of chromogen substrate would bring colour change on enzyme

hydrolysis, linked with 2° antibody.
Sandwich ELISA

Figure depicting sandwich ELISA. Antigen to be measured is sandwiched between primary and
secondary antibody and rest of the process remains same.
SANDWICH ELISA

• Antigen can be detected or measured by a sandwich ELISA.

• In this technique, the antibody (rather than the antigen) is immobilized on a

microtiter well. A sample containing antigen is added and allowed to react with
the immobilized antibody. After the well is washed, a second enzyme-linked
antibody specific for a different epitope on the antigen is added and allowed to
react with the bound antigen.

• After any free second antibody is removed by washing, substrate is added, and
the colored reaction product is measured.
Variations in the enzyme-linked immunosorbent assay (ELISA) technique allow determination of antibody or antigen. Each
assay can be used qualitatively, or quantitatively by comparison with standard curves prepared with known concentrations
of antibody or antigen. Antibody can be determined with an indirect ELISA (a), whereas antigen can be determined with a
sandwich ELISA (b) or competitive ELISA (c). In the competitive ELISA, which is an inhibition-type assay, the concentration
of antigen is inversely proportional to the color produced.
Competitive ELISA
• Competitive ELISA works on the principle that the test antigen and a conjugated
version of the same antigen would compete for the limited number of specific
antibody binding sites pre-coated on ELISA plate.

• This assay could also be done reversibly by the antibody competing for the target
site of the coated antigen.

• The labelled antibody would compete with the native antibody in the sample.

• In a competitive assay, the strength of signal emitted from the assay is inversely
proportional to the concentration of antigen or antibody.
Competitive ELISA

Diagram showing competitive ELISA which provides sensitive variation for detection and measuring of
antigen.
COMPETITIVE ELISA
• Another variation for measuring amounts of antigen is competitive ELISA.

• In this technique, antibody is first incubated in solution with a sample containing

antigen. The antigen-antibody mixture is then added to an antigen coated
microtiter well. The more antigen present in the sample, the less free antibody
will be available to bind to the antigen-coated well. Addition of an enzyme-
conjugated secondary antibody (Ab2) specific for the isotype of the primary
antibody can be used to determine the amount of primary antibody bound to the
well as in an indirect ELISA.

• In the competitive assay, however, the higher the concentration of antigen in the
original sample, the lower the absorbance.
Variations in the enzyme-linked immunosorbent assay (ELISA) technique allow determination of antibody or antigen. Each
assay can be used qualitatively, or quantitatively by comparison with standard curves prepared with known concentrations
of antibody or antigen. Antibody can be determined with an indirect ELISA (a), whereas antigen can be determined with a
sandwich ELISA (b) or competitive ELISA (c).

In the competitive ELISA, which is an inhibition-type assay, the concentration of antigen is inversely proportional to the
color produced.
Comparison of the four types of ELISA methods
Procedure
1. Coating of ELISA Plate with antigen:
• Pipette 50 µl of dilute antigen into each well of the ELISA plate.
• Incubate it overnight at 4°C or at 37°C for 1 hour on plate shaker.
• Wash the plate with washing buffer 3 times. (Invert the plates over blotting paper
and tap the plates to remove residual solutions in the wells)

2. First Reaction:
• Pipette 50 µl of dilute sera to the antigen coated wells in triplicates.
• Incubate at 37°C for 1 hour on plate shaker.
• Wash the plate 3 times with washing buffer and remove residual solution.
3. Second Reaction:
• Pipette 50 µl of diluted anti-mouse IgG-conjugate to the wells.
• Incubate at 37°C for 30 min on plate shaker.
• Wash the plate wells with washing buffer and remove residual solution.

4. Third Reaction:
• Pipette 50 µl of TMB (Tetramethyl benzidine) to each well.
• Incubate at 25°C for 10 min on plate shaker.

5. Reaction Termination:
• Pipette 50 µl of 1 M H2SO4.

6. Absorbance Reading:
• Record the ELISA absorbance reading in ELISA reader at 450 nm.
Observation
• Colour will be observed in the wells of ELISA plate as shown in picture.

Fig. : ELISA Plate Reader

ELISA plate showing colour on completion of reaction. The colour intensity can be measured by ELISA plate reader.
Intensity of the colour is directly proportional to the recorded absorbance.
Calculation
• In general, for calculation of the assay value of ELISA, a standard curve with
absorbance on Y-axis against concentration on X-axis is drawn.

• Then assay value (i.e. the amount of antigen or antibody in the unknown sample)
is estimated/ extrapolated from the absorbance of the sample.

To achieve this, following steps are taken:

• 1. It is important to take ELISA samples in duplicates or in triplicates. This is done
to avoid any handling error as the sample volumes added in the ELISA wells is
very small (100ul) which is prone to error. Once reaction is complete, absobance
is taken for all the samples in ELISA plate reader at a particular optical density
(OD).
• 2. Then calculate the average of the absorbance values (duplicates or triplicates)
for each set of standards and samples.

• 3. Though the negative sample/blank/zero standard appear transparent with

naked eye but it does have some absorbance value. So subtract the average
value of zero standard OD from all the other standards and the test samples (this
step is unnecessary in procedure of competitive ELISA).

• 4. Then create a standard curve and extrapolate the concentration of the

antigen in the test sample using its absorbance value.
• 5. The recorded absorbance values converted to Percentage Inhibition can be
inferred as:
• Positive when the percentage inhibition value is greater than 50%.
• Negative when the percentage inhibition value is lesser than 50%.
Precautions
• 1. In the entire test use proper negative control (without the sera/or without
antigen).

• 2. Personal protective equipment especially using gloves and eye protection gear,
is recommended.

• 3. Avoid cross mixing of reagents.

• 4. Dispense the reagents in the bottom of the well and avoid bubbles.
References
• IGNOU study material
• Kuby- Immunology
Questions?
Thanks