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Immunochemical Methods in the Clinical Laboratory

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Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D., DABCC Mark A. Bowman, Ph.D., MT(ASCP) The University of Florida University of Florida ... – PowerPoint PPT presentation

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Title: Immunochemical Methods in the Clinical Laboratory


1
Immunochemical Methods in the Clinical Laboratory
  • Roger L. Bertholf, Ph.D., DABCC
  • Mark A. Bowman, Ph.D., MT(ASCP)

2
The University of Florida
3
University of Florida Health Science Centers in
Gainesville and Jacksonville
4
The University of Iowa
5
University of Iowa College of Medicine
6
Florida vs. Iowa
7
The American Society of Clinical Pathologists
  • Marie Bass, MT(ASCP)
  • Manager, ASCP Workshops for Laboratory
    Professionals
  • Kathleen Dramisino, MT(ASCP)
  • Workshop coordinator
  • Tommie Ware
  • A/V and materials support

8
Classification of immunochemical methods
  • Particle methods
  • Precipitation
  • Immunodiffusion
  • Immunoelectrophoresis
  • Light scattering
  • Nephelometry
  • Turbidimetry
  • Label methods
  • Non-competitive
  • One-site
  • Two-site
  • Competitive
  • Heterogeneous
  • Homogeneous

9
Properties of the antibody-antigen bond
  • Non-covalent
  • Reversible
  • Intermolecular forces
  • Coulombic interactions (hydrogen bonds)
  • Hydrophobic interactions
  • van der Waals (London) forces
  • Clonal variation

10
Antibody affinity
11
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

12
Factors affecting solubility
  • Size
  • Charge
  • Temperature
  • Solvent ionic strength

13
The precipitin reaction
Precipitate
Zone of equivalence
Antibody/Antigen
14
Single radial immunodiffusion
15
Single radial immunodiffusion
16
Electroimmunodiffusion
  • Why would we want to combine immunodiffusion with
    electrophoresis?
  • SPEED
  • Specificity
  • Carl-Bertil Laurell (Lund University, Sweden)
  • Laurell Technique (coagulation factors)
  • Rocket electrophoresis

17
Electroimmunodiffusion
18
Immunoelectrophoresis
  • Combines serum protein electrophoresis with
    immunometric detection
  • Electrophoresis provides separation
  • Immunoprecipitation provides detection
  • Two related applications
  • Immunoelectrophoresis
  • Immunofixation electrophoresis

19
Immunoelectrophoresis
20
Immunoelectrophoresis
P
C
P
C
P
C
?
?
?
?
?
21
Immunofixation electrophoresis
22
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

23
Light reflection
24
Molecular size and scattering
25
Distribution of scattered radiation
26
Nephelometry vs. Turbidimetry
0-90
27
Rate nephelometry
28
Additional considerations for quantitative
competitive binding immunoassays
  • Response curve
  • Hook effect

29
Competitive immunoassay response curve
Bound label
Antigen concentration
30
Logistic equation
a
c
Bound label
Slope b
d
Log antigen concentration
31
Logit transformation
a
Bound label
d
Log antigen concentration
32
Logit plot
Logit y
Log antigen concentration
33
High dose hook effect
Bound antigen
Antigen concentration
34
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

35
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

36
Radioisotope labels
  • Advantages
  • Flexibility
  • Sensitivity
  • Size
  • Disadvantages
  • Toxicity
  • Shelf life
  • Disposal costs

37
Enzyme labels
  • Advantages
  • Diversity
  • Amplification
  • Versatility
  • Disadvantages
  • Lability
  • Size
  • Heterogeneity

38
Fluorescent labels
  • Advantages
  • Size
  • Specificity
  • Sensitivity
  • Disadvantages
  • Hardware
  • Limited selection
  • Background

39
Chemiluminescent labels
  • Advantages
  • Size
  • Sensitivity
  • S/N
  • Disadvantages
  • Hardware
  • ?

40
Chemiluminescent labels
41
Chemiluminescent labels
42
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

43
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

44
Enzyme-linked immunosorbent assay
45
ELISA (variation 1)
46
ELISA (variation 2)
47
Human anti-animal antibodies
  • Humans exposed to animals can produce antibodies
    to animal immunoglobulins
  • Heterophilic antibodies
  • Anti-isotypic
  • Anti-idiotypic
  • Human anti-mouse antibodies (HAMA) are most
    common
  • Anti-animal antibodies can cross-link capture and
    detection reagent antibodies

48
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

49
Immobilized antibody methods
  • Coated tube
  • Coated bead
  • Solid phase antibody methods

50
Coated tube methods
51
Coated bead methods
52
Microparticle enzyme immunoassay (MEIA)
Glass fiber matrix
53
Magnetic separation methods
54
Magnetic separation methods
55
Electrochemiluminescence immunoassay (Elecsys
system)
56
ASCEND (Biosite Triage)
57
ASCEND
58
ASCEND
59
Solid phase light scattering immunoassay
60
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

61
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

62
Typical design of a homogeneous immunoassay
63
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

64
EMIT method
65
EMIT signal/concentration curve
Signal (enzyme activity)
Antigen concentration
66
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

67
Molecular electronic energy transitions
68
Polarized radiation
69
Fluorescence polarization
Orientation of polarized radiation is maintained!
70
Fluorescence polarization
But. . .
Orientation of polarized radiation is NOT
maintained!
71
Fluorescence polarization immunoassay
72
FPIA signal/concentration curve
Signal (I??/I?)
Antigen concentration
73
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

74
Cloned enzyme donor
Monomer (inactive)
75
Cloned enzyme donor immunoassay
76
CEDIA signal/concentration curve
Signal (enzyme activity)
Antigen concentration
77
Other approaches to homogeneous immunoassay
  • Fluorescence methods
  • Electrochemical methods
  • Enzyme methods
  • Enzyme channeling immunoassay

78
Substrate-labeled fluorescence immunoassay
79
Fluorescence excitation transfer immunoassay
80
Electrochemical differential polarographic
immunoassay
81
Prosthetic group immunoassay
82
Enzyme channeling immunoassay
83
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

84
Molecular imprinting
85
A final thought. . .
In science one tries to tell people, in such a
way as to be understood by everyone, something
that no one ever knew before. But in poetry, it's
the exact opposite.
Paul Adrien Maurice Dirac (1902- 1984)
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