• Immunoassay – two words ; immuno- and assay
Immuno- representing immune, immunity, or immunology
Assay is an investigative (analytic) procedure in laboratory
assessing or quantitatively measuring the presence,
amount, or functional
activity of a target entity (the analyte).
What is immunoassay?
Immunoassay is a biochemical method that uses the
specificity of an antigenantibody reaction to detect and
quantify target molecules in biological samples.
3. The principles of immunoassays
The immunoassay will use those highly specific
antibodies to probe for molecules of interest
when they are in mixtures with other molecules
5. 1. Enzyme immunoassay
• An enzyme immunoassay is any of several
immunoassay methods that use an enzyme
bound to an antigen or antibody. These may
Enzyme-linked immunosorbent assay (ELISA)
6. What is ELISA?
Enzyme-linked immunosorbent assay is a
biomolecular technique that utilizes the
specificity of an antibody, as well as the
sensitivity of enzyme assays, to detect and
quantify molecules such as hormones, peptides,
antibodies, and proteins.
7. • ELISA Components
• • Coated Plates- The 96-well plates are made of polystyrene and coated with
• either inactivated antigen or antibody. This coating is the binding site for the
• antibodies or antigens in the sample
• • Sample Diluent- Most assays require a specific dilution of the sample
• • Controls- Controls are also used to validate the assay and to calculate sample
• • Conjugate- ELISA conjugates are enzyme-labeled antibodies or antigens that
• react specifically to plate-bound sample analytes.
• • Substrate –e.g For peroxidase conjugates, the substrate is a mixture of hydrogen
• peroxide and a chromogen that reacts with the enzyme portion of the conjugate
• to produce color.
• • Wash Concentrate -a buffered solution containing detergent used to wash away
• unbound materials from the plates.
• • Stop Solution- The stop solution stops the enzyme-substrate reaction and,
• thereby, the color development.
10. Methods of ELISA
• Direct ELISA
Refers to an ELISA in which only a labeled
primary antibody is used when the presence
of an antigen is analyzed
11. • Indirect ELISA
Refers to an ELISA in which the antigen is
bound by the primary antibody which then is
detected by a labeled secondary antibody
12. • Sandwich ELISA
The sandwich ELISA quantify antigens
between two layers of antibodies (i.e. capture
and detection antibody).The antigen to be
measured must contain at least two antigenic
epitope capable of binding to antibody
13. • Competitive ELISA
Also known as inhibition ELISA
The central event of competitive ELISA is a
competitive binding process executed by
original antigen (sample antigen) and add-in
14. Applications of ELISA
•detection of Mycobacterium antibodies in tuberculosis
•detection of rotavirus in feces
•detection of hepatitis B markers in serum
•detection of hepatitis C markers in serum
•detection of enterotoxin of E. coli in feces
•detection of HIV antibodies in blood samples
•detection of SARS-CoV-2 antibodies in blood samples
15. 2. RADIOIMMUNOASSAY (RIA)
• It is an immunoassay that uses radiolabeled
molecules in a stepwise formation of
• A RIA is a very sensitive in vitro assay
technique used to measure concentrations of
substances, (for example, hormone levels in
blood) by use of antibodies.
16. 3. Counting Immunoassay (CIA)
• An application of particle counting technology
to serological tests .
• • Latex particles are agglutinated by
antibodies or antigens of interest and are
quantified by scattered laser light while
passing through a controlled sheath flow,
which is also used in flowcytometry.
17. Applications of Counting Immunoassay
• The detection of hepatitis B virus (HBV)
• Anti-adultT-cell leukemia antibodies
• Antitoxoplasma antibodies , hormones , and
serum acute-phase proteins.
18. 4.Chemiluminescence Immunoassay
• Chemiluminescence immunoassay (CLIA) is an
assay that combine chemiluminescence
technique with immunochemical reactions.
• Similar with other labeled immunoassays (RIA,
FIA, ELISA), CLIA utilize chemical probes which
could generate light emission through chemical
reaction to label the antibody.
• CLIA utilize chemical probes which could
generate light emission through chemical
reaction to label the antibody
19. 5. Fluoroimmunoassay (FIA)
• uses as the detection reagent;a fluorescent
compound which absorbs light or energy
(excitation energy) at a specific wavelength
and then emits light or energy at a different
• It is sometimes more sensitive than RIA .