Dr. JITENDRA AGRAWAL
FIRST YEAR RESIDENT
An immunoassay is a specific type of biochemical test that
measures the presence or concentration of a substance
(referred to as the "analyte") in solutions that frequently
contain a complex mixture of substances.
Antibody/Antigen reaction provides the means of
generating a measurable result.
“Immuno” refers to an immune response that causes the
body to generate antibodies.
“Assay” refers to a test.
An immunoassay is a test that uses immunocomplexing
when antibodies and antigens are brought together.
An antibody is a protein produced in the body to a foreign
An antigen is the substance that the body is trying to
eliminate by mounting an immune response.
An analyte is anything measured by a laboratory test.
Immunoassays may measure either the antigen or
Immunoassays use one or more select antibodies to detect
analytes of interest.
All immunoassays require the use of labeled material in
order to measure the amount of antigen or antibody
A label is a molecule that will react as part of the assay,
so a change in signal can be measured in the
Examples of a label
a radioactive compound,
an enzyme that causes a change of color in a solution,
or a substance that produces light.
Categories of Immunoassay
Labels may be applied to either
..or the antigen.
In a competitive format,
unlabeled analyte (usually
the antigen) in the test
sample is measured by its
ability to compete with
the labeled antigen in the
In a competitive
immunoassay, less label
measured in the assay
means more of the
unlabeled (test sample)
antigen is present.
There are two versions of the
• One Step format
• Two step format
One step competitive format
In the one step competitive format , both the labeled antigen
reagent (Ag*) and the unlabeled specimen (or test sample
analyte) compete for a limited amount of antibody.
Two step competitive format
In the two step competitive format, the antibody concentration
of the reaction solution is present in excess in comparison to
the concentration of antigen.
Antibody reagent is first incubated with specimen containing
antigens of interest; then in the second step, labeled antigen is
More sensitive than one step.
Noncompetitive assay formats give
the highest level of sensitivity and
They are normally used to measure
critical analytes such as cardiac and
In noncompetitive assays, the measurement
of the labeled analyte (usually the antibody)
is directly proportional to the amount of
antigen present in the sample.
Enzyme Immunoassay (EIA)
In enzyme immunoassays (EIA), enzyme labels
Typical enzyme labels include alkaline
phosphatase, horseradish peroxidase and galatosidase.
EIA tests typically use a change in color,
emmission of light or other signal.
A sandwich ELISA.
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen present binds to capture
(3) detecting antibody is added, and binds to antigen;
(4) enzyme-linked secondary antibody is added, and to
(5) substrate is added, and is converted by enzyme to
Radioimmunoassay (RIA) techniques were developed in
the 1960s and use radioactive isotopes as a label
Very sensitive in vitro assay technique
Radioactive substances are used
Commonly used radioisotope is I 125
Micro curies of radioactivity – minimum radiation
Micro-gram and Pico-gram quantities of substances can be
Modification of RIA
Antigen attached to plate well – allergen
Radio labeled ligand used will attach only to IgE antibody –
specific for allergen.
Used to detect specific allergen in persons suspected to be
suffering from Type I hypersensitivity.
Fluorescence Polarization Immunoassay (FPIA)
Homogeneous competitive fluoresence immunoassay.
With competitive binding, antigen from the specimen and antigenfluorescein (AgF) labeled reagent compete for binding sites on the
FPIA is used to provide accurate and sensitive measurements of small
toxicological analytes such as therapeutic drugs and drugs of abuse.
Fluorescence Polarization Immunoassay
FPIA uses three concepts to measure specific analytes in a
Rotation of molecules in solution
Fluorescein is a fluorescence label that absorbs light at 490 nm and
releases this energy at 520 nm.
Larger molecules rotate more slowly in solution that smaller
Because of this, we can distinguish between the smaller antigenfluorescein (AgF) label from antibody bound antigen-fluorescein (AbAgF).
Surround Optical Fiber Immunoassay (SOFIA)
in vitro diagnostic platform incorporating a surround
optical fiber assembly that captures fluorescence
emissions from an entire sample.
extremely high limit of detection , sensitivity and
sensitivity is measured at the attogram level (10−18g),
making it approximately one billion times more sensitive
than conventional diagnostic techniques.
Surround Optical Fiber Immunoassay (SOFIA)
ability to detect naturally occurring prions in the blood
and urine of disease carriers
first reliable ante mortem screening test for vCJD, scrapie
and other transmissible spongiform encephalopathies
novel type of diagnostic immunoassay
using magnetic beads as labels
involves the specific binding of an antibody to its antigen,
where a magnetic label is conjugated to one element of
The presence of magnetic beads is then detected by a
magnetic reader (magnetometer) which measures the
magnetic field change induced by the beads.
The signal measured by the magnetometer is
proportional to the analyte (virus, toxin, bacteria, cardiac
marker,etc.) quantity in the initial sample.
Text book of Biochemistry for medical students by DM
Textbook of Microbiology, Annanth narayan.