Applications of Immunochemical Methods in the Clinical Laboratory

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Applications of Immunochemical Methods in the
Clinical Laboratory

• Roger L. Bertholf, Ph.D.
• Associate Professor of Pathology
• University of Florida College of Medicine

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The University of Florida
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University of Florida Health Science Center in
Gainesville
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The University of Florida
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University of Florida Health Science
Center/Jacksonville
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Classification of immunochemical methods

• Particle methods
• Precipitation
• Immunodiffusion
• Immunoelectrophoresis
• Light scattering
• Nephelometry
• Turbidimetry

• Label methods
• Non-competitive
• One-site
• Two-site
• Competitive
• Heterogeneous
• Homogeneous

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Analytical methods using labeled
antigens/antibodies

• What is the function of the label?
• To provide a means by which the free antigens, or
  antigen/antibody complexes can be detected
• The label does not necessarily distinguish
  between free and bound antigens

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Types of labels

• Radioactive
• Enzyme
• Fluorescent
• Chemiluminescent

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Heterogeneous immunoassays

• Competitive
• Antigen excess
• Usually involves labeled competing antigen
• RIA is the prototype

• Non-competitive
• Antibody excess
• Usually involves secondary labeled antibody
• ELISA is the prototype

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The birth of immunoassay

• Rosalyn Yalow and Solomon Berson developed the
  first radioimmunoassay in 1957

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Coated tube methods
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Coated bead methods
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Enzyme-linked immunosorbent assay
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Microparticle enzyme immunoassay (MEIA)
Glass fiber matrix
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Magnetic separation methods
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Magnetic separation methods
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Electrochemiluminescence immunoassay(Elecsys
system)
Flow cell
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ASCEND (Biosite Triage)
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ASCEND
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ASCEND
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Homogeneous immunoassays

• Virtually all homogeneous immunoassays are
  one-site
• Virtually all homogeneous immunoassays are
  competitive
• Virtually all homogeneous immunoassays are
  designed for small antigens
• Therapeutic/abused drugs
• Steroid/peptide hormones

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Typical design of a homogeneous immunoassay
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Enzyme-multiplied immunoassay technique (EMIT)

• Developed by Syva Corporation (Palo Alto, CA) in
  1970s--now owned by Behring Diagnostics
• Offered an alternative to RIA or HPLC for
  measuring therapeutic drugs
• Sparked the widespread use of TDM
• Adaptable to virtually any chemistry analyzer
• Has both quantitative (TDM) and qualitative (DAU)
  applications forensic drug testing is the most
  common use of the EMIT methods

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EMIT method
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EMIT signal/concentration curve
Signal (enzyme activity)
Antigen concentration
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Fluorescence polarization immunoassay (FPIA)

• Developed by Abbott Diagnostics, about the same
  time as the EMIT was developed by Syva
• Like the EMIT, the first applications were for
  therapeutic drugs
• Currently the most widely used method for TDM
• Requires an Abbott instrument

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Molecular electronic energy transitions
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Polarized radiation
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Fluorescence polarization
Orientation of polarized radiation is maintained!
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Fluorescence polarization
But. . .
Orientation of polarized radiation is NOT
maintained!
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Fluorescence polarization immunoassay
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FPIA signal/concentration curve
Signal (I??/I?)
Antigen concentration
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Cloned enzyme donor immunoassay (CEDIA)

• Developed by Microgenics in 1980s (purchased by
  BMC, then divested by Roche)
• Both TDM and DAU applications are available
• Adaptable to any chemistry analyzer
• Currently trails EMIT and FPIA applications in
  market penetration

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Cloned enzyme donor
Monomer (inactive)
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Cloned enzyme donor immunoassay
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Substrate-labeled fluorescence immunoassay
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Fluorescence excitation transfer immunoassay
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Electrochemical differential polarographic
immunoassay
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Prosthetic group immunoassay
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Enzyme channeling immunoassay
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Early theories of antibody formation

• Paul Ehrlich (1854-1915) proposed that antigen
  combined with pre-existing side-chains on cell
  surfaces.
• Ehrlichs theory was the basis for the genetic
  theory of antibody specificity.

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The Template theory of antibody formation

• Karl Landsteiner (1868-1943) was most famous for
  his discovery of the A/B/O blood groups and the
  Rh factor.
• Established that antigenic specificity was based
  on recognition of specific molecular structures
  he called these haptens formed the basis for
  the template theory of antibody formation.

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History of molecular imprinting

• Linus Pauling (1901-1994) first suggested the
  possibility of artificial antibodies in 1940
• Imparted antigen specificity on native globulin
  by denaturation and incubation with antigen.

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Fundamentals of antigen/antibody interaction
CH2-CH2-CH2-CH3
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Molecular imprinting (Step 1)
Methacrylic acid Porogen
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Molecular imprinting (Step 2)
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Molecular imprinting (Step 3)
Cross-linking monomer Initiating reagent
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Molecular imprinting (Step 4)
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Comparison of MIPs and antibodies
Antibodies
MIPs

• In vivo preparation
• Limited stability
• Variable specificity
• General applicability

• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability

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Immunoassays using MIPs

• Therapeutic Drugs Theophylline, Diazepam,
  Morphine, Propranolol, Yohimbine (?2-adrenoceptor
  antagonist)
• Hormones Cortisol, Corticosterone
• Neuropeptides Leu5-enkephalin
• Other Atrazine, Methyl-?-glucoside

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Aptamers
Target
1014-1015 random sequences
Oligonucleotide-Target complex
Unbound oligonucleotides
Target
Aptamer candidates
PCR
New oligonucleotide library
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Thank You!