CLINICAL CHEMISTRY CHAPTER 6

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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 well 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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Immunoelectrophoresis ( IEP )
Step 1. Patient plasma is placed in a well and
undergoes electrophoresis.
Precipitant lines against 3 proteins


Step 2 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 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 doesnt 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 patients ( 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
0
Concentration
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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 )
Enzyme tag
Homogenous technique - no separation step of
antibody - bound and free antigen Steric
hindrance Antibody binding to the
enzymetaggedantigen 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

• 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
Fluorescence Polarization
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
Untagged antigen
Fluorescence Polarization
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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