1 clinical chemistry chapter 6 immunoassays. 2 introduction –in the last chapter, we discussed a...
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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.
+=
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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TOP 10