DISEASES AND DIAGNOSTICS BIOTECHNOLOGY

ELISA – MODULE 5

What is ELISA?

ELISA (enzyme-linked immunosorbent assay)is an analytical biochemical assay used to

study antibodies, glycoproteins, peptides, proteins, and hormones.

A solid-phase enzyme immunoassay (EIA) in this assay is used to detect the targeted protein
in a liquid sample using targeted antibodies. The antigens are attached to the surface, and the
antibodies are applied over it. The specific antibody and antigen bind while the other
unbound antibodies are washed off into the solution.The antibody-antigen reaction is
measured by adding a substrate that produces a colour change, which is used to measure the
analyte concentration in the samples.

ELISA has been used as one of the essential tools in plant pathology, medicine, and
biotechnology and in industries for quality checks of their biological products.

It is carried out in a 96-well plate and can measure and study multiple samples in a single
experiment. It is different from other antibody assays because of its data reproducibility and
quantitative results.

Principle of ELISA

ELISA works on the principle of antigen-antibody interactions. The antibody is a protein

produced by the organism’s immune system and an antigen is a protein coming from any
other foreign source.

In ELISA, special absorbent plates, such as NUNC Immuno plates, are used, which allows
antigens or antibodies to stick to the plate surface. In most cases, antigens are stuck to the
surface and specific antibodies, conjugated with an enzyme or fluorophore, bind to the
antigen. This initiate a reaction that can be detected as a colour change.The more the intensity
of the signal, the more the concentration of the protein. Thus, ELISA is an effective assay to
measure antigen-antibody concentration compared to other immunological assays.
Figure: An illustration of the basic ELISA setup showing how the assay works. [5]

Types of ELISA

ELISA tests are of many types based on the bonding of antigens or antibodies and how they
are used in the process.

1. Direct ELISA

In this assay, antigens or proteins are immobilized on the plate surface. The plate is incubated
with a solution of enzyme-labelled antibody. The specific antibody binds to the specific
antigen and the reaction is observed when a substrate is added, which binds the enzyme-
conjugated antibody and gives a signal. The intensity of the signal is used to measure the
concentration of the analyte in the sample.

Advantages –

 Quick because only one antibody and fewer steps are used.
 Cross-reactivity of secondary antibody is eliminated.

Disadvantages –
  Immunoreactivity of the primary antibody might be adversely affected by labelling
with reporter enzymes or tags.
 Labelling primary antibodies for each specific ELISA system is time-consuming and
expensive.
 Limited number of conjugated primary antibodies available commercially.
 No flexibility in choice of primary antibody label from one experiment to another.
 Minimal signal amplification.

Figure: An illustration of the process of direct ELISA.

2. Indirect ELISA

The indirect ELISA has steps similar to direct ELISA, with an additional amplification
detection step. In the process, analytes or antibodies are immobilized in the wells of the
microplate, which are then incubated with antibodies having the ability to bind to antigens.

The plates are first incubated with primary antibodies followed by secondary antibodies. The
primary antibodies are unconjugated and bind with specific antigens, whereas the secondary
antibodies are conjugated with enzymes or fluorophores and bind to primary
antibodies.Adding the substrate to the plate produces a signal that’s measured to calculate the
amount of antigen bound to the antibodies in the well.

Indirect ELISA is the method of choice to detect the presenceof serum antibodies against
human immunodeficiencyvirus (HIV), the causative agent of AIDS.

Advantages -

 A wide variety of labelled secondary antibodies are available commercially.

 Versatile because many primary antibodies can be made in one species and the same
labelled secondary antibody can be used for detection.
  Maximum immunoreactivity of the primary antibody is retained because it is not
labeled.
 Sensitivity is increased because each primary antibody contains several epitopes that
can be bound by the labeled secondary antibody, allowing for signal amplification.
 Different detection methods can be used with the same primary antibody
(colorimetric, chemiluminescent, etc.).

Disadvantages -

 Cross-reactivity might occur with the secondary antibody, resulting in nonspecific

signal.
 An extra incubation step is required in the procedure.

-OR -

Figure: An illustration of the process of indirect ELISA and the reaction involved.

3. Sandwich ELISA

It is the most common type of ELISA used in labs. In this technique, the antibody (rather
thanthe antigen) is immobilized on a microtiter well. A samplecontaining antigen is added
and allowed to react with theimmobilized antibody.After the well is washed, a second
enzyme-linked antibody specific for a different epitope on theantigen is added and allowed to
react with the bound antigen.After any freesecond antibody is removed by washing,substrate
is added, and the coloured reaction product ismeasured.
 -OR-

Figure: An illustration of the process of sandwich ELISA and the reaction involved.

4. Competitive ELISA

This type of ELISA is used for small molecules.In this process, the antibodies are
immobilized in the wells of the microplate and incubated with antigens. Then, a conjugated
antigen, instead of a conjugated antibody, is used in this process.

The less the target antigen is present in the sample, the more conjugated antigen-antibody
complex, and the more signal will be detected. However, if the concentration of the target
antigen in the sample is higher, the binding of the conjugated antigen and antibody will be
lesser, and the signal detected will be low. This is how the concentration of the targeted
analytes is studied in the desired sample.

Advantages -

 Highly sensitive and highly specific for target antigen as two antibodies are used for
capture and detection.
 Different detection methods can be used with the same capture antibody.

Disadvantages -
  Requires more optimization to identify antibody pairs and to ensure there is limited
cross-reactivity between the capture and detection antibodies.

Figure: An illustration of the process of competitive ELISA and the reactions involved.

ELISA Assay Procedure

Here are the general steps to conducting ELISA experiments. However, it needs to be
tweaked based on the specific experiments:

 Add 100 ul peptide (@4ug/ml) in a coating buffer to wells of the microtiter plate.
 Incubate the plate for 2 hours at 37 ℃ or overnight at 4 ℃.
 Remove the coating solution and wash the plates three times using 100 ul PBS and
0.05%Tween 20.
 Dry the plate on a paper towel.
 Add 100 ul blocking buffer and 3% skim milk in PBS per well to block the remaining
protein binding sites.
 Incubate the plate for one hour at 37 ℃.
  Wash the plate three times with 100 ul PBS-0.05% Tween 20.
 To each well, add 50 ul of diluted antibody.
 Incubate the plates at 37 ℃ with gentle shaking for an hour.
 Wash the plate three times with 100 ul PBS-0.05% Tween 20.
 Add 50 ul of conjugated secondary antibodies. Before use, the antibody should be
diluted in a blocking buffer at the optimal concentration.
 Incubate the plate at 37 ℃ for an hour.
 Wash the plate six times with 100ul PBS and 0.05% Tween20.
 Prepare the substrate solution by mixing TMB (Tetramethylbenzidine), acetic acid,
and 0.03% H2O2 in a ratio of 1:4:5.
 Dispense 50 ul of the substrate solution per well.
 Incubate the plate at 37 ℃ in the dark for 15-30 minutes.
 After sufficient color is developed, add 100 ul stop solution to each well.
 Read the absorbance of the plate.

Advantages and Limitations of ELISA Tests

Advantages:

 Easy to perform: ELISA protocols are simple and easy to follow and require little
hands-on time on experiments.
 High-throughput: In commercial ELISA kits, 96-well plates are usually available.
However, the assay can be easily adapted for 384-well plates.
 High sensitivity and specificity: Antibodies used in ELISAs enable them to detect
antigens at the picogram level in a very specific manner.
 Possibility to test various sample types: ELISA allows you to run multiple samples
at the same time, such as serum, plasma, cellular and tissue extracts, urine, and saliva
among others.
 Quantitative: ELISA is a great tool to determine the concentration of antigens or
analytes in a sample.
 Produce quick results: ELISA is a rapid test thus, yields quick results.

Limitations:
  Temporary readouts: Since enzyme/substrate reactions are responsible for detection
in ELISA tests, readouts must be obtained quickly or in a short span.
 Limited antigen information: The information obtained from ELISA is limited to
the amount or presence of the antigen in the sample.

Applications of ELISA Tests

ELISA tests or assays have extensive applications in life sciences, biotech, and medical
fields. ELISA can be applied to determination of serum antibody concentrations in a virus
test (such as HIV testor West Nile Virus Test). Applications of ELISA have also been
found in home pregnancy test, and in the food industry when detecting potential food
allergens such as milk, peanuts, walnuts, almonds, and eggs. ELISA can also be used in
toxicology as a rapid presumptive screen for certain classes of drugs.Some of them are:

 Diagnosis - In the area of diagnosis, ELISA has proven to be a capable platform

applied worldwide for detecting a variety of disease types in humans and animals. A
number of different commercial ELISA kits are available in the market for detection
of HIV, Influenza, Dengue fever, Ebola, Chagas disease, Leishmaniasis, Lyme
disease, West Nile virus, among others.ELISA is the world’s first screening test for
HIV.
 Vaccine Development - Vaccine development is a very important application of
ELISA. The sera sample from an immunized animal or human model can be tested to
detect the presence of antibodies against certain types of antigens, which were
intentionallyinjected to the host.
 Even in plants pathology, ELISA technique is attracting increasing attention. ELISA
has successfully overcome the drawbacks of the previous serological analyses
performed in phyto-diagnosis.
 Using ELISA, antibodies and antigens can be detected in a sample.
 In a virus test, ELISA is used to measure serum antibodies.
 A rapid ELISA testing kit is used during a disease outbreak to assess the spread of the
disease, e.g. during the recent COVID-19 outbreak.
 Pregnancy Test - A number of different biomolecular entities including human
chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating
hormone (FSH), estriol (E3), and thyrotropin-stimulating hormone (TSH) can be
expressed due to the pregnancy. ELISA can detect some of these proteins from the
 maternal blood, saliva, or urine at the early stages of the pregnancy. HCG is one of
the common hormones that can be detected by ELISA during the first month after
fertilization. Another biomolecule associated with pregnancy is estriol (E3) that can
be detected with ELISA in the saliva at the 6th week of pregnancy.
 Cancer Detection - Highly sensitive detection of cancer provides with the early-stage
diagnostic, which is crucial for patient survival. Cancer biomarkers, however, are
some of the most challenging biomolecular entities as target analytes. Advancements
of ELISA technique have promised its application in detection of cancer biomarkers.

 In the food industry, it is used to detect food allergens, such as peanuts, milk, walnuts,
eggs, and almonds.

Conclusion

ELISA is a biochemical assay used to detect the presence and absence of antigens, proteins,
peptides, glycoproteins, and hormones.

The assay works on the principle of specific binding of antigens with their antibodies. This
interaction is measured by using enzymes that produce a signal based on the amount of
antigen-antibody binding. The four key steps involved in the assay include coating, blocking,
detection, and plate readout.

There are four major types of ELISA – direct, indirect, competitive, and sandwich. All of
these are used in lab workflows based on the experimental goals. The assay has application in
a range of life sciences fields, starting from research studies involving antigen and antibodies
detection, and disease identification, to allergen detection in food samples.