Immunoassays are biochemical methods that use the specificity of antigen-antibody reactions to detect and quantify target molecules in biological samples. The document defines immunoassay and provides details on various types including enzyme immunoassays (ELISA, EMIT), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), and chemiluminescence immunoassay (CLIA). It describes the principles, components, procedures, and applications of each type of immunoassay for detecting molecules like antibodies, hormones, and proteins.

1. IMMUNOASSAYS
Presented by
Aloo Denish
(Biochemist; Microbiologist)
Chuka University; CSI International Ltd
aloo.denish3@gmail.com

2. Definition
Immunoassay – two words ; immuno- and assay
Immuno- representing immune, immunity, or immunology in compound
words
Assay is an investigative (analytic) procedure in laboratory for qualitatively
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 antigen-
antibody reaction to detect and quantify target molecules in biological samples.
The measured entity is often called the analyte, the measurand, or the
target of the assay.
NB-The use of a calibrator is often employed in immunoassays. Calibrators are solutions that are
known to contain the analyte in question, and the concentration of that analyte is generally known.
Comparison of an assay's response to a real sample against the assay's response produced by the
calibrators makes it possible to interpret the signal strength in terms of the presence or
concentration of analyte in the sample.

3. The principles of immunoassays
An immunoassay capitalizes on the specificity of
the antibody-antigen binding found naturally in
the immune system. Antibodies are highly
specific towards particular antigens.The
immunoassay will use those highly specific
antibodies to probe for molecules of interest
when they are in mixtures with other molecules.

4. Types of Immunoassays
•Enzyme Immunoassays (EIA) e.g Enzyme-linked
immunosorbent assays (ELISA)
•Radioimmunoassay (RIA)
•Counting Immunoassay (CIA)
•Fluoroimmnoassay (FIA)
•Chemiluminescenceimmunoassay(CLIA)

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 include:
1.Enzyme-linked immunosorbent assay (ELISA)
2.Enzyme multiplied immunoassay technique
(EMIT)

6. (a) ENZYME-LINKED IMMUNOSORBENT
ASSAYS (ELISA)
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
results.
• 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.

8. ELISA reader

9. Direct ELISA
Refers to an ELISA in which only a labelled primary
antibody is used when the presence of an antigen is
analyzed.

10. Direct ELISA( Continued…)
• A buffered solution of the antigen to be tested for is added to each
well the plate, where it is given time to adhere to the plastic through
charge interactions.
• A solution of nonreacting protein, such as bovine serum albumin or
casein, is added to each well in order to cover any plastic surface in
the well which remains uncoated by the antigen.
• The primary antibody with an attached (conjugated) enzyme is
added, which binds specifically to the test antigen coating the well.
• A substrate for this enzyme is then added. Often, this substrate
changes color upon reaction with the enzyme.
• The higher the concentration of the primary antibody present in the
serum, the stronger the color change. Often, a spectrometer is used
to give quantitative values for color strength.

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. •A sample that must be analyzed for a specific antigen is
adhered to the wells of a microtiter plate
• followed by a solution of nonreacting protein such as bovine
serum albumin to block any areas of the wells not coated
with the antigen.
•The primary antibody,which binds specifically to the antigen,
is then added, followed
•by an enzyme-conjugated secondary antibody.
• A substrate for the enzyme is introduced to quantify the
primary antibody through a color change.
• The concentration of primary antibody present in the serum
directly correlates with the intensity of the color.

13. 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.

14. Sandwich ELISA( Continued…)
• The well surface is prepared to which a known quantity of capture antibody is
bound.
• Any nonspecific binding sites on the surface are blocked.
• The antigen-containing sample is applied to the plate, and captured by antibody.
• The plate is washed to remove unbound antigen.
• A specific antibody is added, and binds to antigen (hence the 'sandwich': the
antigen is stuck between two antibodies).This primary antibody could also be in
the serum of a donor to be tested for reactivity towards the antigen.
• Enzyme-linked secondary antibodies are applied as detection antibodies that also
bind specifically to the antibody's Fc region (nonspecific).
• The plate is washed to remove the unbound antibody-enzyme conjugates.
• A substrate is added to be enzymatically converted into a color or fluorescent or
electrochemical signal.
• The absorbance or fluorescence or electrochemical signal (e.g., current) of the
• plate wells is measured to determine the presence and quantity of antigen

15. 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 antigen.

16. Competitive ELISA( Continued…)
• The primary unlabeled antibody is incubated with the sample antigen
resulting into antibody–antigen complexes
• These antibody/antigen complexes are then added to an antigen-
coated well.The plate is washed, so unbound antibodies are removed.
(The more antigen in the sample, the more Ag-Ab complexes are
formed and so there are less unbound primary antibodies available to
bind to the antigen in the well, hence "competition".)
• The secondary antibody, specific to the primary antibody, is added.This
second antibody is coupled to the enzyme.
• A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal. Absence of color indicates the presence of antigen in
the sample
• The reaction is stopped to prevent eventual saturation of the signal.

17. Reverse ELISA
A fourth ELISA test does not use the traditional wells.This test leaves the antigens
suspended in the test fluid.
• Unlabeled antibody is incubated in the presence of its antigen (sample)
• A sufficient incubation period is provided to allow the antibodies to bind to the antigens.
• The sample is then passed through the Scavenger container.This can be a test tube or a
specifically designed flow through channel.The surface of the Scavenger container or
channel has “Scavenger Antigens” bound to it.These can be identical or sufficiently
similar to the primary antigens that the free antibodies will bind.
• The Scavenger container must have sufficient surface area and sufficient time to allow
the Scavenger Antigens to bind to all the excess Antibodies introduced into the sample.
• The sample, that now contains the tagged and bound antibodies, is passed through a
detector.
• This device can be a flow cytometer or other device that illuminates the tags and
registers the response.

18. Multiple and portable ELISA
Multiple and portable ELISA is a new technique that uses a
multicatcher device with 8 or 12 immunosorbent protruding
pins on a central stick that can be immersed in a collected
sample.The washings and incubation with enzyme-conjugated
antigens and chromogens are performed by dipping the pins in
prefilled microwells with reagents.The main advantage of
these ready-touse lab kits is that they are relatively
inexpensive, can be used for large population screening, and
do not require skilled personnel or laboratory equipment,
making them an ideal tool for low-resource settings

19. Applications of ELISA (1 of 2)
For the detection of HIV antibodies, using competitive Elisa, the wells
of microtiter plate are coated with the HIV antigen.Two specific
antibodies are used, one conjugated with enzyme and the other present
in serum (if serum is positive for the antibody). Cumulative competition
occurs between the two antibodies for the same antigen, causing a
stronger signal to be seen. Sera to be tested are added to these wells
and incubated at 37 °C, and then washed. If antibodies are present, the
antigen-antibody reaction occurs. No antigen is left for the enzyme-
labelled specific HIV antibodies.These antibodies remain free upon
addition and are washed off during washing. Substrate is added, but
there is no enzyme to act on it, so a positive result shows no color
change.

20. Applications of ELISA (2 of 2)
The other uses of ELISA include:
•detection of Mycobacterium antibodies in tuberculosis
•detection of rotavirus in feces
•detection of hepatitis B markers in serum
•detection of hepatitisC 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

21. (b) Enzyme Multiplied Immunoassay
Technique (EMIT)
• It is a common method for qualitative and quantitative determination of
therapeutic and recreational drugs and certain proteins in serum and urine.
• The technique works on the basis that the drug present is proportional to the
inhibition of an enzyme substrate reaction.
• A known quantity of drug is chemically labeled with an enzyme (e.g., glucose-6-
phosphate dehydrogenase), and antibodies specific to that drug bind that drug–
enzyme complex so reducing enzyme activity.
• The introduction of a biological sample containing the same drug will release the
enzyme-labeled drug from the antibody complex, thereby increasing enzymatic
activity.
• Enzyme activity correlates with drug concentration in the specimen as measured
by absorbance changes resulting from the activity of the enzyme on a particular
substrate.
• A change in absorbance is measured by a spectrometer.

22. 2. RADIOIMMUNOASSAY (RIA)
• It is an immunoassay that uses radiolabeled molecules in a stepwise formation of
immune complexes.
• A known quantity of an antigen is made radioactive, frequently by labeling it with
gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine.
• This radiolabeled antigen is then mixed with a known amount of antibody for that
antigen, and as a result, the two specifically bind to one another.
• Then, a sample of serum from a patient containing an unknown quantity of that same
antigen is added.
• This causes the unlabeled (or "cold") antigen from the serum to compete with the
radiolabeled antigen ("hot") for antibody binding sites.
• As the concentration of "cold" antigen is increased, more of it binds to the antibody,
displacing the radiolabeled variant, and reducing the ratio of antibody-bound
radiolabeled antigen to free radiolabeled antigen.
• The bound antigens are then separated and the radioactivity of the free(unbound)
antigen remaining in the supernatant is measured using a gamma counter.[citation
needed]

23. 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 flow cytometry.
•A similar method is the particle counting immunoassay, in
which nonagglutinated particles are counted with the aid of
instrumentation

24. Applications of Counting Immunoassay
Reported applications of this method, most
of which use serum samples, include the
detection of hepatitis B virus (HBV) antigens
or antibodies, anti-adultT-cell leukemia
antibodies , antitoxoplasma antibodies ,
urinary cotinine, hormones , and serum
acute-phase proteins.

25. 4.Chemiluminescence Immunoassay (CLIA)
• Luminescence is the emission of light by a substance when it returns
from an excited state to a ground state i.e. spontaneous emission of
light by a substance not resulting from heat; or "cold light". It is thus a
form of cold-body radiation.
• Chemiluminescence (CL) is defined as the emission of
electromagnetic radiation caused by a chemical reaction to produce
light
• Chemiluminescence immunoassay (CLIA) is an assay that combine
chemiluminescence technique with immunochemical reactions.
• CLIA utilize chemical probes which could generate light emission
through chemical reaction to label the antibody.

26.  CLIA have three different label systems according to the difference of physical
chemistry mechanism of the light emission:
Label Chemical Directly Involved in the Light Emission Reaction
This kind of chemical with special structure can transfer to an excited state through
chemical reaction. Photons would be released when the chemical fell to ground
state from the excited state.The typical chemical is acridinium ester and its
derivatives. Exposure of an acridinium ester label to an alkaline hydrogen peroxide
solution triggers a flash of light.
Enzyme Catalyzed Light Emission Reaction
This type of chemiluminescence utilizes enzymes to label antibody.Technically
speaking, it is an enzyme linked immunoassay that uses luminescent chemical as
substrate instead of chromogen.The most widely used enzymes are horseradish
peroxidase (HRP) and alkaline phosphatase (AP), each has its own luminescent
substrates.
Redox Reaction Mediated Light Emission Reaction
Another CL system is noteworthy because the reagent is regenerated and thus can
be recycled.This system utilizes ruthenium tris-bipyridine (bpy) as label, involves
reaction of Ru(bpy)3
3+ and Ru(bpy)3
+ to produce an excited state of Ru(bpy)3
2+, a
stable species which decays to the ground state by emitting an 620 nm orange

27. 4. 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
wavelength.
•The difference between the wavelength of the
excitation light and the emission light is called the
Stokes Shift.
•It is sometimes more sensitive than RIA

28. References
• https://en.wikipedia.org/wiki/Assay
• https://en.wikipedia.org/wiki/Immunoassay
• https://www.news-medical.net/life-sciences/What-are-Immunoassays.aspx
• https://www.sciencedirect.com/topics/neuroscience/immunoassay
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614608/
• https://www.lornelabs.com/news-events/blog/what-is-chemiluminescent-
immunoassay
• https://en.wikipedia.org/wiki/Radioimmunoassay
• https://www.sciencedirect.com/topics/nursing-and-health-
professions/fluoroimmunoassay
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516346/
aloo.denish3@gmail.com ; Aloo Denish

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