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CLINICAL CHEMISTRYCHAPTER 6

IMMUNOASSAYS

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• Introduction

– In the last chapter, we discussed a variety of analytical techniques

– In this chapter we’ll add some new techniques … They all involve the use of antibody – antigen reactions

– Antibody – Antigen reactions have the advantage of being very specific

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Key Terms

• Antibody• Antigen• Affinity • Avidity• Competitive Immunoassay• Heterogeneous Immunoassay• Homogeneous Immunoassay• IEP• IFE• Nephelometry• Non-competitive Immunoassay• Postzone• Prozone

• Tracer ( Tag )• Turbidimetry• Haptene• Crossreactivity• Polyclonal• Monoclonal• Prozone• Postzone

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• Objectives

– Discuss the basic principles of the following immunoassays

• Immunoelectrophoresis ( IEP )• Immunofixation Electrophoresis ( IFE )• Nephelometry and Turbidimetry• Competitive Immunoassays ( RIA, EIA, FIA )• Non-competitive Immunoassays• Fluorescence Polarization

– Discuss different types of tags or labels used in immunoassays– Classify homogenous, heterogeneous, competitive and

noncompetitive immunoassay techniques– Discuss methods of separation of free and bound tagged reagents

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• Immunoelectrophoresis ( IEP )

– Electrophoresis of antigens is followed by the addition of various antibodies to a parallel trough along the separated proteins

– The antibodies diffuse through the agar and form lines of precipitation with their respective antigens

– The visible precipitant arcs can be compared to known standards to identify specific protein bands – Or to detect missing bands

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Step 2Known anti-sera against one or more proteins isplaced in a parallel trough after electrophoresis anddiffuses through the agar.

Visible lines of precipitation form if antibodyantigen reaction occurs.

Step 1.Patient plasma is placed in a well and undergoes electrophoresis.

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Precipitant lines against 3 proteins

Immunoelectrophoresis ( IEP )

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• IMMUNOFIXATION ELECTROPHORESIS ( IFE )

– Antibody is poured over a completed electrophoresis procedure ( performed on an agar surface ) to produce visible precipitation lines

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• ROCKET ( LAURELL TECHNIQUE )

– Modification of IEP technique

– Antigen ( proteins ) undergo electrophoresis in a supporting agarose gel with specific antibody previously mixed into the gel

– As antigen moves thru the gel , antigen-antibody complexes form creating visible precipitation lines in the shape of long arches or “rockets”

– The length of these “rockets” is proportional to the concentration of antigen

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• Turbidimetry and Nephelometry

– Light is obstructed by insoluble complexes ( usually antibody – antigen )

– Light is obstructed by these insoluble complexes

– Turbidimetry measures transmitted light • Photo-detector is placed at 180 degrees from the light source

– Nephelometry measures scattered light • Photo-detector is placed at 90 degrees from the light source

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• Labeled Immunoassays

– Antigen or antibody is labeled ( tagged ) with a substance that can be detected later on and allows for the detection of an antibody – antigen reaction

– Different binding agents are allowed to attach to substances we want to measure.

– The type of binding agent defines what type of assay it is

• Antibody Immunoassay• Transport Protein Competitive Assay• Hormone receptor Receptor Assay

– Types of tags

• Radioactive isotopes• Enzymes • Fluorescent molecules

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• Competitive Immunoassays

– Competition between tagged and un-tagged antigen for limited antibody

– Tagged antigen Reagent– Untagged antigen Patient antigen we want to measure– Specific antibody Reagent

– Let the competition begin !!!

• Mix the three components together• Allow the antigens to compete for the limited antibody• Antibody will bind with tagged or un-tagged antigen ( it doesn’t

care )• Separation Step : Antibody-Antigen complexes are separated from

free antigen• Tagged antibody-antigen complex is measured

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– The tagged antigen and antibody from the reagent kit are constant.– The only variable is the concentration of the patient antigen

( the thing we want to measure )

– A standard curve can be constructed with known antigen concentrations giving the following general results

• High concentrations of patient antigen means that more of the antibody-antigen complexes are untagged

• Low concentrations of patient antigen means that more of the antibody-antigen complexes are tagged

• There is an inverse relationship between patient antigen concentration and tag activity after the separation process

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Competitive Labeled Immunoassays ( RIA, FIA, EIA )

A competition between tagged antigens ( reagent ) and untagged antigens ( patient )for a limited amount of antibody ( reagent )

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Calculation of RIA / FIA / EIA

The activity of the tag is measured twice :

Before separation step = Total tag activityAfter separation step = Bound tag activity ( antibody – antigen complex )

Note that the separation process removes all unbound ( free ) tag from the testing

% B = B / T x 100

The ratio of the Bound activity to the Total activity ( B / T ) decreases as the concentration of the patient’s ( untagged ) antigen increases.

Using Standard solutions of known antigen concentrations, the % B is plotted againstthe concentrations of the Standards

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Example of an Competitive Assay Standard Curve

B / T

Concentration0

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ELISA ( Enzyme Linked Immunosorbent Assay )

- Antibody is adsorbed onto a solid surface - Tagged and untagged antigens compete for limited antibody- Separation is achieved by pouring off excess unbound ( free ) antigen- Enzymatic activity is inversely proportional to patient antigen concentration

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EMIT ( Enzyme Multiplied Immunoassay Technique )

Homogenous technique - no separation step of antibody - bound and free antigen

Steric hindrance : Antibody binding to the enzyme–tagged–antigen inhibits enzymatic activity

Patient antigen concentration is inversely proportional to enzyme activity

Enzyme tag

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Immunometric Technique

- Immunometric techniques utilize a tagged antibody

- Patient antigen concentration is proportional to measured tag activity

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Fluorescence Polarization Immunoassay

• Competitive Immunoassay

• Homogenous assay – No separation step required

• Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient ) compete for specific antibody in a curvet

• The curvet is exposed to polarized fluorescent light

• Large molecules ( tagged - antigen – antibody complexes ) emit polarized light, where as smaller molecules ( free tagged antigens ) do not

• The amount of polarized light emitted is inversely proportional to the concentration of patient ( untagged ) antigen

• Fluorescence Polarization is used by the ABBOTT TDX analyzer, commonly used for Therapeutic Drug Monitoring ( TDM )

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Example of Polarized and “Normal” Light

- Normal light has wavelengths that occur in all planes

- A polarizing filter blocks all planes except one, but the wavelength is unchanged

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Tagged antigen

Untagged antigen

Most of the limited antibody will bind with fluorescent tagged antigen. Large bound molecules will increase emission of polarized light.

Antibody

( Low concentration of patient antigen )

Fluorescence Polarization

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( High concentration of patient antigen )

Most of the limited antibody will bind with untagged patient antigen.Free unbound fluorescent antigen will decrease emission of polarizedlight.

Fluorescence Polarization

Tagged antigen

Untagged antigenAntibody

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