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8/10/2019 1336336525.4649Labeled Immunoassay Mazen Final (1)
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Labeled Immunoassay
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Immunoassay
An immunoassay is a test that uses antibodyand antigen complexes as a means ofgenerating a measurable result.
An antibody:antigen complex is also known as
an immuno-complex. Immuno refers to animmune response that causes the body togenerate 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 forthe antigen.
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Immunoassay
Both the presence of antigen or antibodies canbe measured.
Example, when detecting infection the presenceof antibody against the pathogen is measured.
For measuring hormones such as insulin, theinsulin acts as the antigen.
For numerical results, the response of the fluidbeing 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 thequantity of the unknown is found from the curve.
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History and Background
In the year 1959, Drs.
Rosalyn Yalow & Soloman
Berson invented the
radioimmunoassay, whichapplied the use of
radioisotopes in the
measurement of insulin.
The RIA is the predecessorof modern immunoassays.
Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay.
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Immunoassay
The first immunoassays were described for the measurement ofinsulin and thyroxine, respectively.
There are now hundreds of immunoassays for scores of analytesincluding:
hormones,
tumor markers,
drugs, antibodies,
and cardiac markers
covering the fields of endocrinology,
oncology,
hematology,
toxicology, serology,
infectious diseases
Developments in antibodies, labels, and automation have resulted inhighly specific and sensitive assays.
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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 specificbinding has taken place.
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Constituents of Labeled assay
For detection of an analyte, the following
are usually a part of the assay:
1. Labeled and nonlabeled analytes
2. Specific antibody
3. Standards or calibrators
4. A method to separate the bound from freecomponents
5. A method for detection of the label.
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1- Labeled analyte
A labeled reactant is used to detect whether or notspecific 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, orenzyme-linked immunosorbent assay [ELISA]),
chemiluminescent (e.g., acridinium ester),
or fluorescent (e.g., fluorscein).
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Methods of coupling indicator labels to
antigen or antibody
Different methods by whichthe indicator label may becoupled to antigen or
antibody. For example, the
radioactive isotope iodineis covalently linked totyrosine residues presenton antibodies and mostantigens.
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Methods of coupling indicator labels to
antigen or antibody
Fluorochromes or enzymes may becoupled to antigens or antibodies usingglutaraldhyde, a bifunctional reagent that
covalently cross links two aminoacidstogether.
Alternatively enzymes can be linked tostreptavidin for use in systems wherebiotin has been attached to either antigenor antibody.
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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.
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The biotin-Streptavidin indicator label
system
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Production of Antibodies
The production of antibodies is an importantprocess in the use of immunoassays becauseit is the antibody-antigen complexes form thebasic.
Antibodies can be called monoclonal orpolyclonal, depending upon the technique usedto produce them.
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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 isinjected 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.
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Monoclonal antibodies
Monoclonal antibodiesproduction result in veryspecific antibodies that bind only to one antigenepitope, which in turn reduces the occurrence offalse positives in the immunoassay
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Mon
oclonalAntibodies
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3- Standards or calibrators
By running a set of calibrators, a calibration curve is setup in the instruments 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 analyteconcentration value, or result, can be determined.
Calibrators are solutions with
known values that establish the
relationship between the amount
of signal produced in the assayand analyte concentration.
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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.
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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 removedfrom solution by the use of a second precipitating
antibody (anti-antibody).
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4- Separation Methods
Currently, most immunoassays use a solid-phase stage for separation.
Numerous substances, such as polystyrene test
tubes, microtiter plates are used for thispurpose.
Antigen or antibody is attached by physicaladsorption , and when specific binding takes
place, complexes remain attached to the solidphase.
This provides a simple way to separate boundand free reactants.
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5- Methods for detection of label
The last step common for all immunoassays isdetection of the labeled analyte.
The method depends on the label; e.g. 125I iseasily detected in a -counter
Enzymes are generally used to producecoloured products from colourless substratesthat can be determined easily in aspectrophotometer or colorimeter.
Automated plate readers are commerciallyavailable which make reading large numbers ofsamples relatively easy.
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Quality Control
It is essential that quality control procedures beestablished.
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 shouldbe run.
This serves as a check on the quality of thereagents to make sure that the label is readilydetectable under current testing conditions.
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Types of Labeled Immunoassays
1- Competitive and Noncompetitive
Immunoassays
2- Homogeneous and Heterogeneous
Immunoassay Methods
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Specific Ab
Ag L antigen- enzyme conjugate
immobilisation surface
L
L
S
P
Enzym. reaction
Product
measurement
L
Incubation
LL
Coating
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.
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Competitive Immunoassays
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Noncompetitive Immunoassays
Noncompetitive(sandwich) immunoassays generallyprovide the highest level of assay sensitivity and specificity.
The reaction mixture typically includes an excess of labeledantibody, 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.
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Noncompetitive Immunoassays
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Homogeneous VS Heterogeneous
Methods
Immunoassay methods that require separation of
bound Ab-Ag* complex are referred to as
heterogeneousimmunoassays.
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.
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Homogeneous VS Heterogeneous
Methods
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Homogeneous immunoassays
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Types of Immunoassays
Radioimmunoassays (RIAs) utilize a
radioactive label
(usually 125I, 3H
or 14C), whichemits radiation that
can be measured
with a beta or
gamma counter.
Within the categories of competitive, noncompetitive,
homogenous, and heterogeneous, there are specific
types, which include:
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Types of Immunoassays Contd
In the Enzyme MultipliedImmunoassay (EMIT), thedrug in the sample andthe drug labeled withG6PD compete forantibody binding sites.
Binding inhibits enzymeactivity, while freeenzyme remains active tointeract with.
Enzymeactivity/absorbance isdirectly proportional todrug concentration.
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Types of Immunoassays Contd
Enzyme linkedimmunosorbant assay(ELISA): competitive,heterogeneous EIA
Reaction components areabsorbed or bound to thesurface of a solid phase,commonly a well of amicrotiter plate
Absorbance is measuredusing a micro-plate reader
Sample absorbance isinversely proportional todrug concentration
http://en.wikipedia.org/wiki/Image:Microtiter_plate.JPG8/10/2019 1336336525.4649Labeled Immunoassay Mazen Final (1)
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Types of Immunoassays Contd
In the FluorescentPolarized 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 analyteconcentration.
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Immunoassay Results
Qualitative Single point calibration at a specific cutoff
Results are either positive or negative; (i.e. aboveor below the cutoff)
Possible false positives; monoclonal antibodiesrestrict this slightly.
Quantitative Provides numeric results that are an estimate of
drug/compound concentration based on the
measurement of labeled analyte in the solution, andtaking into consideration thecompetitive/noncompetitive nature of the device.
In terms of use on drugs, this is sometimescomplicated by possible cross-reactivities.