Download CLINICAL CHEMISTRY CHAPTER 5

CLINICAL CHEMISTRY
CHAPTER 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
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Antibody
Antigen
Affinity
Avidity
Competitive Immunoassay
Heterogeneous Immunoassay
Homogeneous Immunoassay
IEP
IFE
Nephelometry
Non-competitive Immunoassay
Postzone
Prozone
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Tracer ( Tag )
Turbidimetry
Haptene
Crossreactivity
Polyclonal
Monoclonal
Prozone
Postzone
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• Objectives
– Discuss the basic principles of the following immunoassays
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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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Immunoelectrophoresis ( IEP )
Step 1.
Patient plasma is placed in a well and undergoes electrophoresis.
Precipitant lines against 3 proteins
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Step 2
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Known anti-sera against one or more proteins is
placed in a parallel trough after electrophoresis and
diffuses through the agar.
Visible lines of precipitation form if antibody
antigen reaction occurs.
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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
• Transport Protein
• Hormone receptor
Immunoassay
Competitive Assay
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
– Untagged antigen
– Specific antibody
Reagent
Patient antigen we want to measure
Reagent
– Let the competition begin !!!
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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 activity
After 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 against
the concentrations of the Standards
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Example of an Competitive Assay Standard Curve
B/T
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Concentration
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ELISA ( Enzyme Linked Immunosorbent Assay )
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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 )
Enzyme tag
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
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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
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Competitive Immunoassay
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Homogenous assay – No separation step required
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Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient )
compete for specific antibody in a curvet
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The curvet is exposed to polarized fluorescent light
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Large molecules ( tagged - antigen – antibody complexes ) emit polarized
light, where as smaller molecules ( free tagged antigens ) do not
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The amount of polarized light emitted is inversely proportional to the
concentration of patient ( untagged ) antigen
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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
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Normal light has wavelengths that occur in all planes
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A polarizing filter blocks all planes except one, but the wavelength is unchanged
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Fluorescence Polarization
Tagged antigen
Untagged antigen
Antibody
( Low concentration of patient antigen )
Most of the limited antibody will bind with fluorescent
tagged antigen. Large bound molecules will increase
emission of polarized light.
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Tagged antigen
Fluorescence Polarization
Untagged antigen
Antibody
( High concentration of patient antigen )
Most of the limited antibody will bind with untagged patient antigen.
Free unbound fluorescent antigen will decrease emission of polarized
light.
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