Labeled Immunoassay
Immunoassay
• An immunoassay is a test that uses antibody
and antigen complexes as a means of
generating a measurable result.
• An antibody:antigen complex is also known as
an immuno-complex. “Immuno” refers to an
immune response that causes the body to
generate antibodies, and “assay” refers to a test.
• The assay takes advantage of the specific
binding of an antibody to its antigen.
• The antibodies used must have a high affinity for
the antigen.
Immunoassay
• Both the presence of antigen or antibodies can
be measured.
• Example, when detecting infection the presence
of antibody against the pathogen is measured.
• For measuring hormones such as insulin, the
insulin acts as the antigen.
• For numerical results, the response of the fluid
being measured must be compared to standards
of a known concentration.
• This is usually done through the plotting of a
standard curve on a graph paper, and then the
quantity of the unknown is found from the curve.
History and Background
•
•
In the year 1959, Drs.
Rosalyn Yalow & Soloman
Berson invented the
radioimmunoassay, which
applied the use of
radioisotopes in the
measurement of insulin.
The RIA is the predecessor
of modern immunoassays.
Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay.
Immunoassay
• The first immunoassays were described for the measurement of
insulin and thyroxine, respectively.
• There are now hundreds of immunoassays for scores of analytes
including:
•
•
•
•
hormones,
tumor markers,
drugs, antibodies,
and cardiac markers
• covering the fields of
•
•
•
•
•
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endocrinology,
oncology,
hematology,
toxicology,
serology,
infectious diseases
• Developments in antibodies, labels, and automation have resulted in
highly specific and sensitive assays.
Labeled immunoassays
• Labeled immunoassays are designed for
antigens and antibodies that may be
• small in size
• or present in very low concentrations.
• The presence of such antigens or antibody
is determined indirectly by using a labeled
reactant to detect whether or not specific
binding has taken place.
Constituents of Labeled assay
•
For detection of an analyte, the following
are usually a part of the assay:
1.
2.
3.
4.
Labeled and nonlabeled analytes
Specific antibody
Standards or calibrators
A method to separate the bound from free
components
5. A method for detection of the label.
1- Labeled analyte
• A labeled reactant is used to detect whether or not
specific binding has taken place.
• The label used in immunoassay:
• must not alter the reactivity of the molecule,
• and it should remain stable for the shelf life of the reagent.
• Labels attached to analytes and antibodies may be:
• radioactive, usually iodine-125 (radioimmunoassay and
immunoradiometric assays),
• enzymes such as alkaline phosphatase and horseradish
peroxidase, (enzyme immunoassay or immunometric assay, or
enzyme-linked immunosorbent assay [ELISA]),
• chemiluminescent (e.g., acridinium ester),
• or fluorescent (e.g., fluorscein).
Methods of coupling indicator labels to
antigen or antibody
• Different methods by which
the indicator label may be
coupled to antigen or
antibody.
• For example, the
radioactive isotope iodine
is covalently linked to
tyrosine residues present
on antibodies and most
antigens.
Methods of coupling indicator labels to
antigen or antibody
• Fluorochromes or enzymes may be
coupled to antigens or antibodies using
glutaraldhyde, a bifunctional reagent that
covalently cross links two aminoacids
together.
• Alternatively enzymes can be linked to
streptavidin for use in systems where
biotin has been attached to either antigen
or antibody.
The biotin-Streptavidin/ Avidin indicator
label system
• Biotin is a vitamin that can bind tightly to
either avidin or streptavidin.
• Avidin & streptavidin are proteins.
• The natural attraction of these two proteins
for one another is a property that has been
exploited to facilitate coupling of indicator
molecules to antigens or antibodies.
The biotin-Streptavidin indicator label
system
Production of Antibodies
• The production of antibodies is an important
process in the use of immunoassays because
it is the antibody-antigen complexes form the
basic.
• Antibodies can be called monoclonal or
polyclonal, depending upon the technique used
to produce them.
Polyclonal antibodies
• Polyclonal antibodies may be produced in mammals such as
rabbits or sheep.
• When a foreign substance enters the body, it stimulates the immune
system to produce antibodies to the substance.
• Using this natural reaction, an analyte in as pure form as possible is
injected into the animal stimulating the production of antibodies.
• Antiserum usually contains a mixture of antibodies that recognize
and bind to the same antigen, but they may attach to different
epitopes.
Monoclonal antibodies
• Monoclonal antibodies production result in very
specific antibodies that bind only to one antigen
epitope, which in turn reduces the occurrence of
false positives in the immunoassay
Monoclonal Antibodies
3- Standards or calibrators
• Calibrators are solutions with
known values that establish the
relationship between the amount
of signal produced in the assay
and analyte concentration.
• By running a set of calibrators, a calibration curve is set
up in the instrument’s software and correlates certain
values of signal to known analyte concentrations.
• By comparing levels of signal produced by patient
samples to this calibration curve, a patient analyte
concentration value, or result, can be determined.
4- Separation Methods
• In most assays, once the reaction between antigen and
antibody has taken place, there must a way of
separating reacted from unreacted analyte.
• This can be accomplished by several different means.
Unreacted analyte can be removed by:
• Adsorption on particles such as dextran-coated charcoal,
– These adsorb out the smaller unbound molecules, which
are then separated from bound molecules by
centrifugation or filtration.
– The amount of label remaining in the supernatant
provides an indirect measure of analyte present in the
patient's sample.
4- Separation Methods
• Another means of separation involves precipitation
of antigen-antibody complexes.
– Complexes can be precipitated by adding concentrated
solutions of ammonium sulfate, or ethanol
• Antigen-antibody complexes can also be removed
from solution by the use of a second precipitating
antibody (anti-antibody).
4- Separation Methods
• Currently, most immunoassays use a solidphase stage for separation.
• Numerous substances, such as polystyrene test
tubes, microtiter plates are used for this
purpose.
• Antigen or antibody is attached by physical
adsorption , and when specific binding takes
place, complexes remain attached to the solid
phase.
• This provides a simple way to separate bound
and free reactants.
5- Methods for detection of label
• The last step common for all immunoassays is
detection of the labeled analyte.
• The method depends on the label; e.g. 125I is
easily detected in a γ-counter
• Enzymes are generally used to produce
coloured products from colourless substrates
that can be determined easily in a
spectrophotometer or colorimeter.
• Automated plate readers are commercially
available which make reading large numbers of
samples relatively easy.
Quality Control
• It is essential that quality control procedures be
established.
• This is done to limit random errors, such as
• temperature fluctuations,
• minor changes in the concentration of reagents,
• and changes in detector efficiency.
• A negative control and a positive control should
be run.
• This serves as a check on the quality of the
reagents to make sure that the label is readily
detectable under current testing conditions.
Types of Labeled Immunoassays
1- Competitive and Noncompetitive
Immunoassays
2- Homogeneous and Heterogeneous
Immunoassay Methods
Competitive Immunoassays
In competitive formats, unlabelled analyte (usually antigen) in the test sample is
measured by its ability to compete with labeled antigen in the immunoassay.
immobilisation surface
Specific Ab
antigen- enzyme conjugate
L
Ag
Coating
Incubation
L
L
L
L
L
S
Enzym. reaction
P
Product
measurement
Competitive Immunoassays
Noncompetitive Immunoassays
• Noncompetitive (sandwich) immunoassays generally
provide the highest level of assay sensitivity and specificity.
• The reaction mixture typically includes an excess of labeled
antibody, so that all metabolite is bound.
• The amount of antibody-antigen complex is then measured
to determine the amount of analyte present in the sample.
• The labeled antibody, is directly proportional to the amount
of antigen present in the sample.
Noncompetitive Immunoassays
Homogeneous VS Heterogeneous
Methods
• Immunoassay methods that require separation of
bound Ab-Ag* complex are referred to as
heterogeneous immunoassays.
• Those that do not require separation are referred to as
homogeneous immunoassays.
• Homogeneous methods have been generally applied
to the measurement of small analytes such as abused
and therapeutic drugs.
• Since homogeneous methods do not require the
separation of the bound Ab-Ag* from the free Ag*, they
are generally much easier and faster to perform.
Homogeneous VS Heterogeneous
Methods
Homogeneous immunoassays
Types of Immunoassays
• Within the categories of competitive, noncompetitive,
homogenous, and heterogeneous, there are specific
types, which include:
• Radioimmunoassa
ys (RIAs) utilize a
radioactive label
(usually 125I, 3H
or 14C), which
emits radiation that
can be measured
with a beta or
gamma counter.
Types of Immunoassays Cont’d
• In the Enzyme Multiplied
Immunoassay (EMIT), the
drug in the sample and
the drug labeled with
G6PD compete for
antibody binding sites.
• Binding inhibits enzyme
activity, while free
enzyme remains active to
interact with.
• Enzyme
activity/absorbance is
directly proportional to
drug concentration.
Types of Immunoassays Cont’d
• Enzyme linked
immunosorbant assay
(ELISA): competitive,
heterogeneous EIA
• Reaction components are
absorbed or bound to the
surface of a solid phase,
commonly a well of a
microtiter plate
• Absorbance is measured
using a micro-plate reader
• Sample absorbance is
inversely proportional to
drug concentration
Types of Immunoassays Cont’d
•
In the Fluorescent
Polarized Immunoassay,
the drug in the sample
competes with
fluorescein-labeled drug
for antibody binding
sites.
•
Reaction mixture is
excited by
planepolarized light.
•
As the tracer returns to a
lower energy state, it
emits light; polarization
is measured.
• The polarization value of the sample is
inversely proportional to analyte
concentration.
Immunoassay Results
• Qualitative
– Single point calibration at a specific cutoff
– Results are either ‘positive’ or ‘negative’; (i.e. above
or below the cutoff)
– Possible false positives; monoclonal antibodies
restrict this slightly.
• Quantitative
– Provides numeric results that are an estimate of
drug/compound concentration based on the
measurement of labeled analyte in the solution, and
taking into consideration the
competitive/noncompetitive nature of the device.
– In terms of use on drugs, this is sometimes
complicated by possible cross-reactivities.