IMMUNOASSAYS

PRINCIPLE OF IMMUNOASSAY
• Immunoassay is a test that uses antibodies and antigen complexes
as a
means of generating a measurable result.
• An antibody: antigen complex is also known as an immune
complex.
• An immunoassay is a test that utilizes immune complexing when
antibodies
and antigens are brought together
 TYPES OF IMMUNOASSAY

• COMPETITIVE IMMUNOASSAY

• NON COMPETITIVE IMMUNOASSAY

• HOMOGENOUS IMMUNOASSAY
• HETEROGENEOUS IMMUNOASSAYS
 COMPETITIVE IMMUNOASSAY

• In a competitive format , unable analyte (usually the

antigen) in the test sample is measured by its ability to
compete with the labelled antigen in the immunoassay
• Its is less label measured in the means more of the
unlabelled( test sample)antigen is present.
 NON COMPETITIVE IMMUNOASSAY

• In non-competitve assay , the measurement of the labelled

anlayte usually the antibody) is directly proportional to the
amount of antigen present in the sample
• Noncompetitive assay formats can use either one step or two
step methods.
• In the two step assays format , there are wash steps in which
the sandwich binding complex is isolate and wash to remove
excess unbound labelled reagent
 ONE-SITE, NONCOMPETITIVE
IMMUNOASSAYS

• The unknown analyte in the sample binds with labelled

antibodies.
• The unbound, labelled antibodies are washed away, and the
bound, labelled antibodies are measured
• The intensity of the signal is directly proportional to the
amount of unknown analyte
 TWO-SITE, NONCOMPETITIVE
IMMUNOASSAYS
• The analyte in the unknown sample is bound to the antibody site,
then the labelled antibody is bound to the analyte.
• The amount of labelled antibody on the site is then measured.
• It will be directly proportional to the concentration of the analyte
because the labelled antibody will not bind if the analyte is not
present in the unknown sample.
• This type of immunoassay is also known as a sandwich assay as
the analyte is "sandwiched" between two antibodies.
 HOMOGENEOUS IMMUNOASSAYS

• Homogenous methods have been generally applied to the

measurement of small analytes such as abused and therapeutics
drugs.
• since homogenous methods do not require the separation of the
bound Ab-Ag* from the free Ag*, there are generally much easier
and faster to perform homogeneous assay.
 HETROGENOUS IMMUNOASSAY
• As in a competitive, heterogeneous immunoassay, unlabelled
analyte in a sample competes with labelled analyte to bind an
antibody the labelled, unbound analyte is separated or washed
away, and the remaining labelled, bound analyte is measured.
 PRODUCTION OF ANTIBODIES

• An antibody is a protein that is produced by the body in

response to an invading (foreign) substance.
• Antibodies are produced as part of the body’s immune response
to protect itself.
• Antibodies (Ab) are a type of protein called immunoglobulin.
• The most common one is immunoglobulin G(IgG).
• IgG is a protein composed of two main structural and functional
regions
 TYPES OF ANTIBODIES
There are two types of antibodies
• Polyclonalantibodies
• Monoclonal antibodies
•Polyclonal antibodies recognize multiple sites on antigens, or
monoclonal antibodies recognize single sites on antigens.
 SEPARATION OF BOUND AND
UNBOUND DRUG
•In competitive immunoassays, the amount of antigen is directly
proportional to the amount of signal
•Homogenous immunoassays do not require separation of unbound
complexes from the bound complexes, and thus are faster and easier
to perform then heterogeneous immunoassays.
 TECHNIQUES OF IMMUNOASSAY
• RADIOIMMUNOASSAY
• OPTICAL IMMUNOASSAY
• ENZYME IMMUNOASSAY
• ELISA(ELISA(ENZYME-LINKEDIMMUNOBSORBENT ASSAY)
• FLUOROIMMUNOASSAY
• LUMINESCENCE IMMUNOASSAY
 RADIOIMMUNOASSAY
• Radioimmunoassay (RIA) involves the separation of a
protein (from a mixture) using the specificity of antibody -
antigen binding and quantitation using radioactivity
 THE TECHNIQUE :
• A mixture is prepared of radioactive antigen
• Because of the ease with which iodine atoms can be
introduced into tyrosine residues in a protein, the radioactive
isotopes 125I or 131I are often used.
•antibodies against that antigen.
•Known amounts of unlabeled ("cold") antigen are added to
samples of the mixture. These compete for the binding sites
of the antibodies.
• At increasing concentrations of unlabeled antigen, an increasing
amount of radioactive antigen is displaced from the antibody
molecules.
• The antibody-bound antigen is separated from the free antigen in the
supernatant fluid, and
• The radioactivity of each is measured
•The main drawbacks to radioimmunoassay are the expense and hazards
if preparing and handling the radioactive antigen.
• Both 125I or 131I emit gamma radiation that requires special counting
equipment
• The body concentrates iodine atoms radioactive or not in the thyroid
gland where they are incorporated in thyroxine (T4).
 ADVANTAGES OF RIA
•Highly specific
•Highly sensitivity
•Possible to detect picograms of Ag
•Sepharose beads used in RIA are reuseable
 DISADVANTAGE OF RIA
•Radiation hazards : uses radiolabel led reagents
•Requires specially trained persons
•Labs require special license to handle radioactive material
•Requires special arrangements for
•Requisition
•Storage of radioactive material
•Radioactive waste disposal
 APPLICATIONS OF RIA
•Analysis of hormones, vitamins, metabolites, diagnostic markers
•ACTH,FSH,T3,T4,Glucagon,insulin,testosterone,vitamin ,
,prostaglandins, glucocorticoids
THERAPEUTIC DRUG MONITORING
•Barbiturates,morphine,digoxin
•Diagnostic procedures for detecting infection
•HIV, Hepatitis A,B
•Diagnosis of allergy
 OPTICAL IMMUNOASSAY
SURROUND OPTICAL FIBER
IMMUNOASSAY (SOFIA)
• An ultra-sensitive,
• 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 dynamic
range.
• Sensitivity is measured at the attogram level (10−18g),making it
approximately one billion times more sensitive than conventional
diagnostic techniques.
• 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
 MAGNETIC IMMUNOASSAY
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 pair.
• 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.
 ENZYME IMMUNOASSAY(EIA)
• In enzyme immunoassays (EIA), enzyme labels are used. Typical
enzyme labels include alkaline phosphatase, horseradish peroxidase
and galatosidase.
• EIA tests typically use a change in color, emission of light or other
signal.
ELISA(ENZYME-LINKED IMMUNOSORBENT
ASSAY)
BASIC PRINCIPLE OF ELISA:
• Use an enzyme to detect the binding of antigen (Ag)antibody (Ab).
• The enzyme converts a colorless substrate(chromogen) to a colored
product, indicating the presence of Ag : Ab binding.
• An ELISA can be used to detect either the presence of Antigens or
antibodies in a sample depending how the test is designed.
• ELISA was developed in 1970 and became rapidly accepted
 ELISA QUALITATIVE/QUANTITATIVE
•Qualitative determines antigen or antibody is present or absent
•Quantitative determines the quantity of the antibody
• The highest dilution of the specimen usually serum which gives a
positive reaction in the test 
TYPES OF ELISA:
• A number of variations of ELISA have been developed, allowing
qualitative detection or quantitative measurement of either antigen or
antibody.
1. INDIRECT ELISA: The indirect ELISA detects the presence of
antibody in a sample. The antigen for which the sample must be
analyzed is adhered to the wells of the microliter plate.
•The primary antibody present in the sample bind specifically to the
antigen after addition of sample.
•The solution is washed to remove unbound antibodies and then enzyme
conjugated secondary antibodies are added. The substrate for enzyme is
added 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.
 2. SANDWICH ELISA:

The sandwich ELISA is used to identify a specific sample antigen.

The wells of microliter plate are coated with the antibodies.
•Non-specific binding sites are blocked using bovine serum albumin.
The antigen containing sample is applied to the wells. A specific
primary antibody is then added after washing.
• This sandwiches the antigen. Enzyme linked secondary antibody is
added that binds primary antibody.
•Unbound antibody-enzyme conjugates are washed off. The
substrate for enzyme is introduced to quantify the antigens.
3. COMPETITIVE ELISA:
•This type of ELISA depends on the competitive reaction between
the sample antigen and antigen bound to the wells of microliter plate
with the primary antibody.
• First, the primary antibody is incubated with the sample. This
results in the formation of Ag-Ab complex which are then added to
the wells that have been coated with the same antigens.
After an incubation, unbound antibodies are washed off. The more
antigen in the sample, more primary antibody will bind to the sample
antigen. Therefore there will be smaller amount of primary antibody
available to bind to the antigen coated on well.
•Secondary antibody conjugated to an enzyme is added, followed by
a substrate to elicit a chromogenic signal. Concentration of color is
inversely proportional to the amount of antigen present in the
sample.
 ELISA (ENZYME-LINKED
IMMUNOSORBENT ASSAY)
•ELISA is a widely-used method for measuring the concentration
of a particular molecule (e.g., a hormone or drug) in a fluid such
as serum or urine. It is also known as enzyme immunoassay or
EIA.
• ELISA has many of the advantages (e.g., sensitivity, ease of
handling multiple samples) without the disadvantages of dealing
with radioactivity (like in RIA).
THE TEST REQUIRES:
•The antibodies fixed to a solid surface, such as the inner surface of a
test tube;
•a preparation of the same antibodies coupled to an enzyme.
•This is one (e.g., β-galactosidase) that produces a colored product
from a colorless substrate.
PERFORMING THE TEST:
•The tubes are filled with the antigen solution (e.g., urine) to be
assayed. Any antigen molecules present bind to the immobilized
antibody molecules.
• The antibody-enzyme conjugate is added to the reaction mixture.
The antibody part of the conjugate binds to any antigen molecules
that were bound previously, creating an antibody-antigen-antibody
"sandwich".
• After washing away any unbound conjugate, the substrate
solution is added.
•After a set interval, the reaction is stopped (e.g., by adding 1 N
NaOH) and the concentration of colored product formed is
measured in a spectrophotometer.
•The intensity of color is proportional to the concentration of
bound antigen.
APPLICATIONS OF ELISA :
•To evaluate either the presence of antigen or the presence of
antibody in a sample.
• it is a useful tool for determining serum antibody concentrations
(such as with the HIV test or West Nile virus).
•It has also found applications in the food industry in detecting
potential food allergens, such as milk, peanuts, walnuts, almonds,
and eggs and as serological blood test for coeliac disease.
•ELISA can also be used in toxicology as a rapid presumptive screen
for certain classes of drugs. detection of Mycobacterium antibodies
in tuberculosis.
 FLUOROIMMUNOASSAY

• In a fluoroimmunoassay the antibodies are labeled with fluorescent

probes.
•After incubation with antigen the antibody-antigen complexes are
isolated and the fluorescent intensity is measured. 
• Radioisotopes and enzymes were replaced by new fluorescent
compound in labeling the antibodies and antigen.
• Substances used such as chelate of Europium which fluoresces
when it excite by light in the presence of a developing solution
• Emission duration is short but less than that of the background
noise. can be measured by special instrument which is
unfortunately very expensive.
• It is 10 times more sensitive than RIA.
• A modern fluorescent based immunoassay 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.

FACTORS IMPACTING IMMUNOASSAYS

•Accuracy and precision
•Calibration and control
FLUORESCENCE IMMUNOASSAY:
• In fluorescence, a photon of an appropriate energy excites the
molecule from its ground state to a higher electronic state.
• When the molecule returns to the ground state, energy is released
as light emitted at a longer wavelength.
• The difference between the excitation wavelength and the
emission wavelength is the stocks shift.
• A large stokes shift in nanometer means that there is a large
difference between the excitation and emission wavelengths
FOR EXAMPLE,
• Fluorescein, a common flurophore has small stocks shift at 30 min.
It has a maximal absorption at 490nm and emission at 520 nm.
• In contrast a rare earth chelate such as europium has a large stocks
shift of 270 nm.
• It has a maximal absorption at 340 nm and an emission at 610 nm.
•In principle, a fluoroimmunoassay using flurometry has an
advantage over an enzyme immunoassay using colorimetry
• In colorimetry, the light absorbed by a sample is related directly to
the concentration of the absorbing molecule and is independent of
the intensity.
• In fluorometry the intensity of the fluorescence emission is directly
proportional to the intensity of the incident light.
• There are problems associated with fluorescence measurements
1) Endogenous fluorophores , such as bilirubin and proteins, can
increase the non specific background fluorescence and reduce the
sensitivity of FIA.
2) Light scattering by high concentrations of protein, lipid and other
particles in serum will reduce the fluorescence signal.
3) The inner filter effect of hemoglobin and albumin will absorb part of
the excitation or emission beam.
4) Quenching duo to the non specific binding of albumin and interaction
with other specific quenching species may change the quantum yield
of the fluorescence.
An important function of this step is the removal of endogenous
fluorescent compounds and interfering substances from the sample prior
to the detection step.
• This also allows large sample sizes to be used and improves sensitivity
as well as sensitivity.
• Solid phase FIA is a convenient approach either as competitive FIA or
sandwich immunometric assay
FLUORESCENCE POLARIZATION ASSAY
• Fluorescence Polarization (FP) assays are homogeneous, single-step
assays ideally suited for high-throughput screening (HTS) of large
numbers of samples.
• All FP assays employ a large molecular species, or binding partner
(BP), in conjunction with a small, low molecular weight fluorophore-
labeled analyte (FA).
• When the large BP molecule is an antibody, the assay is referred to as
a fluorescence polarization immunoassay (FPIA)
• FPIAs are based on the competition of FA with free (i.e. unlabeled)
analyte in the samples or standards for the high affinity binding site an
antibody.
• A micro plate well filled with the FA: antibody complex will give a
high FP reading.
• Addition of a increasing amounts of unlabeled analyte will result in a
competition between the unlabeled FA for the antibody
•As the competition happens, some of the FA will be released from the
antibody, and will resume its intrinsic, rapid rate of rotation. This will
cause a detectable loss of FP in the well.
• The addition of large amount of analyte will result in a much larger
reduction in the mP of the well (well Plotting mP versus analyte
concentration allows the construction of a standard curve with a broad
dynamic range.
•This is similar to, but not strictly analagous to, the sigmoidal dose-
response curve in a traditional solid phase EIA
 LUMINESCENCE IMMUNOASSAY

• Luminescence is described as the emission of light from a substance

as it returns from an electronically excited state to ground state.
• The various forms of luminescence (bioluminescence,
chemiluminescence, photoluminescence) differ in the way the excited
state is reached.
FOR EXAMPLE,
•Photoluminescence is simply fluorescence; the excitation is initiated
by light at a particular wavelength.
•Bioluminescence is characterized by the use of a bioluminescent
compound, such as luciferin and firefly luciferase.
•Chemiluminescence is light produced by a chemical reaction
PRINCIPLE:
• Luminescence means that light is emitted by a substance when it
returns from an excited state to a ground state.
• There are different types of luminescence and the various forms
differ in the way that they achieve the excited state.
• For chemiluminescence it is light produced by a chemical reaction.
This chemiluminescent substance can be excited by an oxidation
reaction forming an intermediate.
• It is when this immediate return to a stable ground state happens,
that a photon is released and this is detected by the luminescent
signal instrument.
• The particular luminescence indicates the presence of the antigen.
The amount of the particular biological molecule, which is being
looked for and is present in the sample, is based on the
luminescence observed.
• There are different types of substrate that are used for
chemiluminescence, with the most popular types being luminol or
its derivatives. 
TYPES OF LUMINESCENCE  
Excitation event Process

Chemicals Luminol, Isoluminol, Chemiluminescence

acridinium ester

Biochemical Luciferin, aequorin Bioluminescence

Electromagnetic Ruthenium, Tris Electroluminescence

(bipyridly) chelate

Photons inorganic phosphors Photoluminescence

Chemiluminescent immunoassays are variations of the standard
ELISA where an enzyme converts a substrate to a reaction product
that emits photons of light instead of developing a visible color.
• The chemiluminescent substance is excited by the oxidation and
catalysis forming intermediates.
• When the excited intermediates return back to their stable ground
state, a photon is released, which is detected by the luminescent
signal instrument. CHEMILUMINESCENCE 
•Emission of light with limited emission of heat (luminescence), as
the result of a chemical reaction.
[A] + [b] → [◊] → [products] + light
[A], [b]: reactants
[◊]: Excited intermediate
For example, if [A] is luminol and [B] is hydrogen peroxide in the
presence of a suitable catalyst we have:
Luminol + H2O2 →3-APA[◊] →3-APA + light
Where:
•3-APA is 3-aminophthalate
•3-APA[◊] is the excited state producing light as it decays to a lower
energy level. 
Both bioluminescence and chemiluminescence are widely used for
immunoassays as “luminescence”.
• Luminescent assays are very sensitive and have a wide dynamic
range.
• It is believed that luminescence is the most sensitive detection
method currently in use due to the ability of signal multiplication
and amplification.
• Luminescent reactions are measured in Relative Light Units
(RLU) that are typically proportionate to the amount of analyte
present in a sample
Application of Chemiluninescence
•Chemiluminesence immunoassay
•DNA hybridization detection
•Western blotting
•Forensic science
•Food analysis
 REFERENCES
•Wild, David (Ed.). (2005).The Immunoassay Handbook. Kidlington, Oxford:
Elsevier. 

• Evans, Susan (2004, June 15). Retrieved January 19, 2008, from SACB Online
Web site: http://sacb.org.sg/.

• https://en.m.Wikipedia.org/wiki/Immunoassay