INTRODUCTION TO LABELED IMMUNOASSAY

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LABELEDIMMUNO ASSAY

BY; JOHN ALFREY D. PUEBLO


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INTRODUCTION

Clinical Laboratory must haven a Rapid, specific,and sensitive assay.

LI designed for antigens and antibodies that maybe small in size or present in very lowconcentrations.

Analyte- a substance to be measured

Helps in rapid monitoring and diagnosis of

numerous disease prompt treatment formany such conditions


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Immunoassays are tests that use

antibody and antigen complexes (alsocalled immunocomplexes) to measurethe presence of a specific analyte in asample.

Antibodiesare proteins that are normallyproduced by the immune system inresponse to a foreign substance.

Antigensare the molecules that

antibodies bind to, which in the bodycould be an invading pathogen, or theforeign molecules injected into an animalto trigger the immune response.


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CHARACTERISTICSOF LABELEDIMMUNOASSAYS


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COMPETITIVE VS.

NONCOMPETITIVE ASSAYS

CURRENT TECHNIQUES

1. Fluorescent2. Radioactive

3. Chemiluminescent

4. Enzyme labels
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COMPETITIVE IMMUNOASSAY

unlabelled analyte(usually antigen) inthe test sample is measured by itsability to compete with labeled

antigen in the immunoassay. The unlabeled antigen blocks the

ability of the labeled antigen to bind

because that binding site on theantibody is already occupied.


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The amount of bound label is inversely

proportional to the concentration oflabeled antigen

More label detected, less patientsantigen

Thus, in a competitive immunoassay,less label measured in the assaymeans more of the unlabeled (test

sample) antigen is present. Theamount of antigen in the test sample isinversely related to the amount oflabel measured in the


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competitive format (Figure 1-7).


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One step competitive format

In the one step competitive format (see Figure 1-8),both the labeled antigen reagent (Ag*) and the

unlabeled specimen (or test sample analyte)

compete for a limited amount of antibody.


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Two step competitive format

the antibody concentration of the reaction solutionis present in excess in comparison to theconcentration of antigen.

Antibody reagent is first incubated with specimen

containing antigens of interest second step, labeled antigen is added.

Remember that in the competitive format, lessbound labeled antigen indicates more antigenpresent in the test sample.

Two step competitive assay formats provide severalfold improved assay sensitivity compared to onestep assay formats.


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NON-COMPETITIVEIMMUNOASSAY


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Capture antibody

The amount of label is

directly proportional to theamount of patient antigen


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Unknown patient antigen is then

allowed to react with and becaptured by antibody.After washing, a second antibody with

label is added to the reaction.


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Noncompetitive assay formats can also

utilize either one step or two step methods,as with the competitive assay.

The two step assay format employs washsteps in which the sandwich binding

complex is isolated andwashed to remove excess unboundlabeled reagent and any other interferingsubstances.

The two step noncompetitiveformatusually offers the highest specificity andsensitivity of all the assay formats


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Noncompetitive assayformats

generally provide the highest level ofassay sensitivity and specificity andare applied to the measurement ofcritical analytes such as cardiac andhepatitis markers.

This format is referred to as asandwichassay because analyte isbound (sandwiched) between twohighly specific antibody reagents(Figure 1-10).


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Homogeneous and Heterogeneous

Immunoassay MethodsImmunoassay methods thatrequire separation of bound

Ab-Ag* complexare referredto as heterogeneousimmunoassays.

Those that do notrequireseparation are referred to ashomogeneousimmunoassays.


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Homogeneous methodshave

been generally applied to themeasurement of small analytessuch as abused and therapeuticdrugs.

Since homogeneous methods donot require the separation of thebound Ab-Ag* from the free Ag*,

they are generally much easierand fasterto perform.


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ANTIBODIES

Essential to have high affinity for the antigen.

Competitive binding sites

Random interaction between individual Agand Ab molecules

THE Higher the affinity of antibody for antigen,the larger the amount of antigen bound toantibody and the more accurately specificbinding can be measured.

Ab used should also be very specific forAginvolved in the reaction.

Monoclonal Ab---leads to highly specificantibodies to increase the detection of a mallamount of analyte with great accuracy


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STANDARD OR CALIBRATORS

Known concentrations of a

substance to be measured.Used to established a relationship

between the labeled analytemeasured and unlabeled analyte

that might be present in patientspecimen.


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SEPARATION METHODSeparating reacted from

unreacted analyte.

Solid-phase vehicle forseparation.

Polystyrene test tubes, microtiterplates, glass or polystyrene beads,magnetic beads and cellulosemembrane.

Great carte must be observe.


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DETECTION OF LABEL

The last step common for allimmunoassays is detection of thelabeled analyte.The method depends on the label;

Enzymes are generally used toproduce coloured products fromcolourless substrates that can bedetermined easily in aspectrophotometer or colorimeter.

Automated plate readers arecommercially available which makereading large numbers of samplesrelatively easy.


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QUALITY CONTROL

It is essential that quality controlprocedures be established.

This is done to limit random errors, such astemperature fluctuations,

minor changes in the concentration of reagents,and changes in detector efficiency.

A negative control and a positive controlshould be run.

This serves as a check on the quality of thereagents to make sure that the label isreadily detectable under current testingconditions.

INTRODUCTION TO LABELED IMMUNOASSAY

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