Exercise 6 Enzyme Linked Immunosorbent Assay (ELISA)

EXERCISE 6
ENZYME LINKED
IMMUNOSORBENT ASSAY
(ELISA)
ELISA)

Structure
6.1 Introduction 6.5 Observation
Objectives 6.6 Precautions
6.2 Materials Required 6.7 Terminal Questions
6.3 Principle
6.4 Procedure

6.1 INTRODUCTION
ELISA is a sensitive immunochemical technique for the detection and
quantification of hormones, peptides, proteins and antigens. It is widely utilized
in medicine as diagnostics, 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.

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.

6.2 MATERIALS REQUIRED

• ELISA plate (Fig. 6.1),

• Antigen,

• standard polyclonal anti-serum/antigen-specific monoclonal antibody, 43

BZYEL-142 Immunology: Laboratory
• 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 in 500 ml Distilled Water

8 g Sodium chloride + 0.2 g Potassium chloride + 1.15 g Disodium hydrogen

phosphate + 0.2 g Potassium dihydrogen phosphate

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 in distilled water1.21 g Tris in 50 ml and adjust the pH to 7.6 with

HCl.

Make up the final volume to 100 [Link] 10 times before Use.

ELISA Substrate

1. Diaminobenzidine (DAB):

Dissolve 6 mg Diaminebenzidine 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-phenyldiamine in 100 ml of

0.1M Sodium citrate Buffer (pH 5.0)

44 Add 50 µl of Hydrogen peroxide [just before use]

Exercise 6 Enzyme Linked Immunosorbent Assay (ELISA)

Fig. 6.1: ELISA plate.

6.3 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)
followed by 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:

• 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 (Fig.
6.2).

Antibody-Enzyme conjugate Colour change

Chromogen

Antibody-Enzyme binds
Test Antigen to Antigen

Fig. 6.2: Figure depicting the direct [Link] the detection of antigen in a given
biological sample, enzyme (e.g. Horse reddish peroxidase) conjugated
antibody is used. When antigen is detected, chromogenic susbstrate
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 45
BZYEL-142 Immunology: Laboratory
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 (Fig. 6.3)

1° Antibody-antigen 2° Antibody-Enzyme
binding binds to 1° Antibody Chromogen Colour change

Antigen

Fig. 6.3: 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 secondary
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).

• Sandwich ELISA

In sandwich ELISA, the antigen is sandwiched between 1 and 2 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 detection
antibody or secondary antibody,which is enzyme- conjugated is then
introduced to bind with the antigen trapped on the 1° [Link] addition of
chromogen substrate would bring colour change on enzyme hydrolysis, linked
with 2° antibody (Fig. 6.4).

Chromogen
2° Antibody-
Enzyme linked 2°
1° Antibody- Enzyme binds to
Antigen Antibody Colour change
antigen binding antigen

Immobilized
1° Antibody

Fig. 6.4: Figure depicting sandwhich ELISA. Antigen to be measured is

sandwiched between primary and secondary antibody and rest of the
process remains same as previously explained.

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
46 coated antigen. The labelled antibody would compete with the native antibody
Exercise 6 Enzyme Linked Immunosorbent Assay (ELISA)
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 (Fig.
6.5).

Sample & conjugated Chromogen

1° Antibody Sample antigen
Conjugated antigen antigen competes for 1°Ab Colour change
coat

Fig. 6.5: Diagram showing competitive ELISA which provides sensitive variation
for detection and measuring of antigen.

You can refer to Table 6.1 for better understanding of the differences among
four types of ELISA.

Table 6.1: Comparison of the four types of ELISA methods.

Direct ELISA Indirect ELISA Sandwich Competitive

ELISA ELISA
1 Fastest ELISA Economical – High specificity – No requirement
test –less steps, less involvement of for sample
reagents and requirement of detection and processing
less error- prone the labelled capture
antibody. antibodies.
2 High background Likelihood of Cross-reactivity Cross-reactivity
noise – antigen background gives may occur-
immobilization is noise from background antibody
not specific. No secondary noise too optimization is
cross- reactivity antibody. between difficult for this
captured and assay
detection
antibodies.
3 Low flexibility - High flexibility- High flexibility – Most flexible –
specific antibody a wide range of since both direct since direct,
for specific 1° primary and indirect indirect and
antigen. antibody can assay can be sandwich ELISA
be used for a deployed. can be deployed
single labelled
2°- antibody.
4 Assay choice for Assay choice Assay of choice Assay of choice
analyzing the for for analysing for detection of
immune determination complex antigen antigens that are
response to an of antibody sample not bound to 2
antigen concentration. different
antibodies.
47
BZYEL-142 Immunology: Laboratory
5 Absence of High sensitivity 2-5 times higher Less sensitive
signal – signal sensitivity than but more robust
amplification – amplification direct and
less sensitive indirect ELISA

6.4 PROCEDURE
1. Coating of ELISA Plate with antigen

Pipette 50 µl of dilute antigen into each of wells of the ELISA plate

Incubate it overnight @ 4 °C or @ 37 °C for 1 h on plate shaker

Wash the plate with washing buffer 3 times

[Invert the plates over plotting 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

[Maintain triplicates]

Incubate @ 37 °C for 1 h on plate shaker

Wash the plate 3 times with washing buffer and remove residual solution

3. Second Reaction

Pipette 50 µl of diluted antimouse IgG-conjugate to the wells

Incubate @ 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 wells

Incubate @ 25 °C for 10 min on plate shaker

5. Reaction Termination: Pipette 50 µl of 1 M H2SO4(Sulphuric acid)

6. Absorbance Reading: Record the ELISA absorbance reading at 450

nm.
48
Exercise 6 Enzyme Linked Immunosorbent Assay (ELISA)
6.5 OBSERVATION
You will observe colour on ELISA plate as shown in Fig. 6.6.

Fig. 6.6: ELISA plate showing colour on completion of reaction. The colour
intensity can be measured by ELISA plate reader (See Fig. 6.7).
Intensity of the colour is directly proportional to the recorded
absorbance.

Fig. 6.7: ELISA Plate Reader.

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 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 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 49
BZYEL-142 Immunology: Laboratory
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 inhibiton value is lesser than 50%.

Perent inhibition can
range from O to
100%. It depends
6.6 PRECAUTIONS
upon the
concentiation of the 1. In the entire test use proper negative control (Without the sera/or without
substrate and antigen).
enzyme. When the
inhibitor is bound, the 2. Personal protective equipment especially using gloves and eye
substrate can’t bind, protection gear, is recommended.
but when the inhibitor
dissociates, the 3. Avoid cross mixing of reagents.
substrate can bind
4. Dispense the reagents in the bottom of the well, and avoid bubbles.
and be converted to
product.

6.7 TERMINAL QUESTIONS

1. What is the role of antibody-linked to enzyme Horse Radish Peroxidase
(HRP) in the experiment?

(a) Antigen

(b) Primary antibody

(c) Secondary antibody

(d) None of the above

2. The color intensity of spot is ………………. the concentration of antigen

in the sample.

(a) Independent of

(b) Directly proportional to

(c) Inversely proportional to

(d) None of the above

3. Compare direct ELISA and indirect ELISA.

4. Briefly describe the principle of ELISA.

5. In ELISA, proteins in a sample are adsorbed to an inert surface, usually

a 96-well polystyrene plate. Why surface is washed with a solution of an
50 inexpensive nonspecific protein-like, casein?
Exercise 6 Enzyme Linked Immunosorbent Assay (ELISA)
6. A patient was given an antibody, and his serum developed Ag/Ab
complex. Can we measure the serum antibodies of the antibodies
administered? Describe the procedure involved and the type of ELISA to
be used.

Acknowledgement of Figures
Illustration are drawn/Photographs are clicked by the authors of this Exercise.

51