This presentation explores the principles of immunoassays, emphasizing the vital role of antibodies in diagnostics. It covers antigen-antibody interactions, assay formats, and detection methods, including ELISAs, fluorescence immunoassays, and lateral flow assays. Factors influencing immunoassay performance, such as antibody specificity and sensitivity, are discussed.

1. Immunoassay Principles
BY GIDEON ETOWA

2. An Immunoassay is a chemical test that uses an antibody
and antigen to form an immunological reaction that helps
to detect or quantify a specific substance.
It’s highly sensitive and specific because antibodies are
usually specific for certain antigens.
Immunoassays can detect antigens in extremely low
concentrations that standard tests cannot detect.

3. An immunological
reaction involves
binding an antibody
to a specific antigen
to create an immune
complex commonly
referred to as an
antigen-antibody
complex.

4. An antibody with an affinity for a
specific antigen will bind with that
antigen to form an antigen-antibody
complex.
The formed complex is then used to
quantify or qualify the concentration
of the antigen.
Principle of
Immunoassay

5. Antibodies
Antibodies are proteins produced by the immune
system in response to a specific antigen (foreign
substances such as bacteria, viruses, and toxins) in the
blood.
Monoclonal antibodies only bind to one specific
antigen. These antibodies tend to be very expensive
as they give more specific and accurate results.
Polyclonal antibodies, on the other hand, can bind to
more than one antigen and as such, they’re less
expensive and less specific for antigens.

6. Immunoassay
techniques

7. Enzyme-linked immunosorbent assay
(ELISA)
In the ELISA technique, an antibody labeled with an enzyme
forms an antigen-antibody complex with the antigen (or
antibody) in the sample. A substrate is then added to the
complex and on the catalysis of the substrate (usually a
chromogen), the reaction produces a color change that is read
colorimetrically.

8. ELISA is a solid phase immunoassay
performed in microtiter well plates of
usually 96 wells that are precoated with
the capture antibody. It can be either
qualitative or quantitative.
Basically, it involves, immobilizing antigens
on a solid surface, forming of
immunocomplex and treating the enzyme-
linked antibody with a substrate, and then
measuring the reaction colorimetrically.

9. Chemiluminescence Immunoassay (CHLIA)
Chemiluminescence involves the emission of light by a
chemical reaction, so instead of producing heat, the reaction
gives off light. In CHLIA the antibody is linked with an enzyme
and when a substrate is added to the immune complex, its
breakdown is catalyzed by the enzyme, and the product is
detected by the emission of a photon instead of a particular
color as is obtainable in ELISA.

10. The chemiluminescent substance (usually
luminol or its derivatives), is excited and
oxidized by the reaction, and when it
returns to its solid ground state, that is
when a light signal is produced. The
amount of light signal produced is then
used to quantify the concentration of the
antigen of interest.

11. Electrochemiluminescence Immunoassay (ECLIA)
It differs from the CLIA in the sense that voltage is required to
trigger the chemiluminescence process.
When a voltage is applied to an electrode, the electrode
surface becomes excited and an electron transfer is produced
between molecules, and the resulting emitted light is
measured.

12. In the ECLIA process, ruthenium and TPA
are excited by voltage and TPA reduces
ruthenium and returns it to its ground
state. It’s in this process that light is
emitted.
The cycle repeats for as long as the
voltage is applied, and this results in an
amplification of the light signal which is
equivalent to the concentration of the
analyte.

13. “Electro” = electrical stimulation.
“Chem” = chemical reaction.
“Luminescence” = produces light
Electrochemiluminescence (ECL)
The ECL technology
Antigen
Antibody
Magnetic field Magnetic field
Procell
Ruthenium
Biotin
Voltage

14. Immunoassay
types

15. Sandwich
In sandwich immunoassay, two antibodies are needed in the
immunocomplex. A capture antibody and a detection antibody.
The target antigen contains two antigenic sites to allow for the
binding of the capture and detection antibodies.
The capture antibody can be a polyclonal antibody that has a high
affinity for the antigen of interest. The detection antibody is
usually enzyme-linked or contains a chemiluminescent agent to aid
in the detection of the analyte and its quantification.

17. Competitive
Here the patient’s unlabeled antigen competes with a labeled
antigen for a limited amount of antibodies.
Because the two antigens compete for the same antibody, the
labeled antigen must react in the same way as the unlabeled
antigen.

18. The amount of bound, labeled
antigen-antibody complex in
equilibrium is inversely
proportional to the amount of
the analyte of interest present
in the specimen.

19. Bridging
Bridging ELISA is a type of sandwich ELISA where a dimeric or
oligomeric antigen (usually an antibody in a sample) is
detected by a capture and detection antibody. The antigen
connects the two specific antibodies.
In this type, the antigen is usually an antibody to a drug, and
this method is the industry standard for pharmacokinetic or
ADA (anti-drug antibody testing).

20. The drug is labeled separately
with different tags and any
anti-drug antibodies present
in a sample will form a bridge
between the two labeled
molecules.

21. Electrochemiluminescence (ECL)
technology

22. Thanks for listening