Immunochemical%20Methods%20in%20the%20Clinical%20Laboratory

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Immunochemical Methods in the Clinical Laboratory

• Roger L. Bertholf, Ph.D., DABCC
• Chief of Clinical Chemistry Toxicology,
  UFHSC/Jacksonville
• Associate Professor of Pathology, University of
  Florida College of Medicine

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Name The Antigen
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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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Aminobenzene Sulphonate, a Hapten
NH2
NH2
NH2
SO3
SO3
SO3
Ortho
Meta
Para
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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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Properties of the antibody-antigen bond

• Non-covalent
• Reversible
• Intermolecular forces
• Coulombic interactions (hydrogen bonds)
• Hydrophobic interactions
• van der Waals (London) forces
• Clonal variation

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Antibody affinity
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Precipitation of antibody/antigen complexes

• Detection of the antibody/antigen complex depends
  on precipitation
• No label is involved
• Many precipitation methods are qualitative, but
  there are quantitative applications, too

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Factors affecting solubility

• Size
• Charge
• Temperature
• Solvent ionic strength

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The precipitin reaction
Precipitate
Zone of equivalence
Antibody/Antigen
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Single radial immunodiffusion
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Single radial immunodiffusion
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Double immunodiffusion
Örjan Ouchterlony
Developed double immunodiffusion technique in 1948
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Double immunodiffusion (Ouchterlony)
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Quantitative double immunodiffusion
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Electroimmunodiffusion

• Why would we want to combine immunodiffusion with
  electrophoresis?
• SPEED
• Specificity
• Carl-Bertil Laurell (Lund University, Sweden)
• Laurell Technique (coagulation factors)
• Rocket electrophoresis

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Electroimmunodiffusion
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Immunoelectrophoresis

• Combines serum protein electrophoresis with
  immunometric detection
• Electrophoresis provides separation
• Immunoprecipitation provides detection
• Two related applications
• Immunoelectrophoresis
• Immunofixation electrophoresis

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Immunoelectrophoresis
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Immunoelectrophoresis
P
C
P
C
P
C
?
?
?
?
?
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Immunofixation electrophoresis
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Particle methods involving soluble complexes

• The key physical property is still size
• Measurement is based on how the large
  antibody/antigen complexes interact with light
• The fundamental principle upon which the
  measurement is made is light scattering
• Two analytical methods are based on light
  scattering Nephelometry and Turbidimetry

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Light reflection
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Molecular size and scattering
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Distribution of scattered radiation
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Nephelometry vs. Turbidimetry
0-90
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Rate nephelometry
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Additional considerations for quantitative
competitive binding immunoassays

• Response curve
• Hook effect

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Competitive immunoassay response curve
Bound label
Antigen concentration
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Logistic equation
a
c
Bound label
Slope b
d
Log antigen concentration
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Logit transformation
a
Bound label
d
Log antigen concentration
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Logit plot
Logit y
Log antigen concentration
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High dose hook effect
Bound antigen
Antigen concentration
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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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Analytical methods using labeled
antigens/antibodies

• What are desirable properties of labels?
• Easily attached to antigen/antibody
• Easily measured, with high S/N
• Does not interfere with antibody/antigen reaction
• Inexpensive/economical/non-toxic

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The birth of immunoassay

• Rosalyn Yalow (1921-) and Solomon Berson
  described the first radioimmunoassay in 1957.

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Radioisotope labels

• Advantages
• Flexibility
• Sensitivity
• Size

• Disadvantages
• Toxicity
• Shelf life
• Disposal costs

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Enzyme labels

• Advantages
• Diversity
• Amplification
• Versatility

• Disadvantages
• Lability
• Size
• Heterogeneity

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Fluorescent labels

• Advantages
• Size
• Specificity
• Sensitivity

• Disadvantages
• Hardware
• Limited selection
• Background

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Chemiluminescent labels

• Advantages
• Size
• Sensitivity
• S/N

• Disadvantages
• Hardware
• ?

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Chemiluminescent labels
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Chemiluminescent labels
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Introduction to Heterogeneous Immunoassay

• What is the distinguishing feature of
  heterogeneous immunoassays?
• They require separation of bound and free ligands
• Do heterogeneous methods have any advantage(s)
  over homogeneous methods?
• Yes
• What are they?
• Sensitivity
• Specificity

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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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Enzyme-linked immunosorbent assay
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ELISA (variation 1)
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ELISA (variation 2)
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Automated heterogeneous immunoassays

• The ELISA can be automated
• The separation step is key in the design of
  automated heterogeneous immunoassays
• Approaches to automated separation
• immobilized antibodies
• capture/filtration
• magnetic separation

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Immobilized antibody methods

• Coated tube
• Coated bead
• Solid phase antibody methods

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Coated tube methods
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Coated bead methods
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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)
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ASCEND (Biosite Triage)
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ASCEND
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ASCEND
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Solid phase light scattering immunoassay
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Introduction to Homogeneous Immunoassay

• What is the distinguishing feature of homogeneous
  immunoassays?
• They do not require separation of bound and free
  ligands
• Do homogeneous methods have any advantage(s) over
  heterogeneous methods?
• Yes
• What are they?
• Speed
• Adaptability

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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
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Fluorescence polarization immunoassay (FPIA)

• Developed by Abbott Diagnostics, about the same
  time as the EMIT was developed by Syva
• Roche marketed FPIA methods for the Cobas FARA
  analyzer, but not have a significant impact on
  the market
• 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
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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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CEDIA signal/concentration curve
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Other approaches to homogeneous immunoassay

• Fluorescence methods
• Electrochemical methods
• Enzyme methods
• Enzyme channeling 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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Artificial antibodies

• Immunoglobulins have a limited shelf life
• Always require refrigeration
• Denaturation affects affinity, avidity
• Can we create more stable artificial
  antibodies?
• Molecular recognition molecules
• Molecular imprinting

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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