1336336525.4649Labeled Immunoassay Mazen Final (1)

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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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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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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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.JPG


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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.

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1336336525.4649Labeled Immunoassay Mazen Final (1)