The document discusses the Enzyme-Linked Immunosorbent Assay (ELISA) technique, which is a biochemical assay used in immunology to detect substances like peptides, proteins, antibodies, and hormones. ELISA uses antibodies and color changing enzymes to identify these molecules in a sample. There are different variations of ELISA including direct, indirect, and double antibody sandwich ELISA. ELISA is a sensitive, cost-effective technique that can test large numbers of samples for antigens or antibodies.

1. Enzyme linked
Immuno-Sorbent Assay
(ELISA)
(Principles and Protocols)

2. What is ELISA ?
Enzyme Linked Immuno-Sorbent Assay (ELISA) is a biochemical
assay technique or Serology based detection used mainly in
Immunology.
It is a plate-based assay designed for detecting and quantifying
substances such as peptides, proteins, antibodies & hormones.
Advantages :
1.Sensitivity of virus diagnosis.
2.Cost effective.
3.A large number of samples can be tested for virus infection.
Disadvantages :
Detection of virus of unknown origin is not possible by serology as
well as its inability to detect antigenic property is not functional
for viroid (absence of coat protein).

3. ELISA :
 Sensitive and efficient
 Good for large scale testing
 Ready to use test-kits available commercially
 Dependent on good antisera (expensive)
Negative result Positive result Microtiter well
Fig : ELISA
plate

4. Fig : ELISA Detector

5. Principles of ELISA :
 The sensitivity of detection depends on amplification signal
during the analytic reaction. In some enzymatic reaction, the
signal generated by enzyme which are linked to the detection
reagents in fixed proportions to allow accurate quantification
(enzyme linked).
 There are two main variations on ELISA method is :
1. ELISA can be used to detect the presence of antigens that are
recognized by an antibody or
2. It can be used to test for antibodies that recognize an antigen.

6. Types of ELISA :
 Qualitative ELISA :
Positive (+ ve) or Negative (- ve ) results.
 Quantitative ELISA :
Optical density or fluorescent units of the sample is
interpolated into a standard curve, which is typically a serial
dilution of the target.

7. ELISA Methods :
 Direct ELISA
 Indirect ELISA
 Double Antibody Sandwich ELISA ( DAS )

8. Ag = Antigen

9. Direct ELISA DAS ELISA Indirect ELISA

10. Antibody Structure :
Antibodies are immune system-related
proteins called immunoglobulins. Each
antibody consist of 4 polypeptide chains and
2 light chains joined to form a ‘ Y ’ shaped
molecules.
The amino acid sequence in the tips of the ‘
Y ‘ varies greatly among different antibodies.
This variable region, composed of 110-130
amino acids, give the antibody its specificity
for antigen binding.
The variable region includes the ends of the
light and heavy chains.
The constant region determines the
mechanism used to destroy antigen.
Antibodies are divided into 5 major classes :
IgM, IgE, IgG, IgA and IgD based on their
constant region structure and immune
function.

11. Antigen(Ag)-Antibody(Ab)
Reaction :

12. Antibodies :
Polyclonal Antibody Monoclonal Antibody
1.Derived from different B-
lymphocyte cell lines.
1.Derived from a single B-
cell clone.
2.Batch to Batch variation
affecting Ab reactivity &
treatment.
2.No Batch to Batch
variations. Effectiveness of
Ab is much more
predictable.
3.NOT Powerful tools for
clinical diagnostic tests.
3.Enable the development of
secure immunoassay
systems.

13. Direct ELISA :
 Protocols :
Direct ELISA is suitable for the detection of proteinaceous antigens and
may require pre-purifications of sample. Direct ELISA can be performed when
desired antibody is available in a pre-conjugated state (fluorometric,
colorimetric, enzymatic).
 Advantages :
1. Fast
2. Eliminates possible non-specific binding of secondary antibody
 Disadvanatges :
1. Reactivity of primary antibody may be reduced conjugation
2. Little signal amplification

14. Indirect ELISA :
 Protocol :
If the primary antibody is not conjugated, then indirect ELISA is required in
which a conjugated secondary antibody is targeted to the isotype ( e.g. mouse
IgG1, goat IgM, chicken IgY etc.) of the primary antibody.
 Advantages :
1. Wide variety of secondary conjugates are available for detection.
2. Immuno-reactivity of the primary antibody is not compromised.
3. Multiple binding of the secondary affords some signal amplification.
 Disadvantages :
1. Extra step in the protocol.
2. Some non-specific binding of the secondary may cause high risk.

15. Double Antibody Sandwich (DAS) ELISA :
Add Antibody in the microtiter well
A. Add Sample / virus + ve
B. Incubation and Washing
C. Addition of Enzyme labelled antibody
D. Incubation and Washing
E. Addition of Substrate ( Hydrogen peroxide+
Diamino benzene)
G. Incubation
H. Brown color in virus + samples

16. Fig : Comparison of Different types of ELISA