Transcript Immunochemical Methods in the Clinical Laboratory

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 Health Science
Centers in Gainesville and Jacksonville
The University of Iowa
University of Iowa College of Medicine
Florida vs. Iowa
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
Classification of immunochemical
methods
• Particle methods
– Precipitation
• Immunodiffusion
• Immunoelectrophoresis
– Light scattering
• Nephelometry
• Turbidimetry
• Label methods
– Non-competitive
• One-site
• Two-site
– Competitive
• Heterogeneous
• Homogeneous
Properties of the antibody-antigen
bond
• Non-covalent
• Reversible
• Intermolecular forces
– Coulombic interactions (hydrogen bonds)
– Hydrophobic interactions
– van der Waals (London) forces
• Clonal variation
Antibody affinity
Ab  Ag  Ab  Ag
[ Ab  Ag ]
Ka 
[ Ab][ Ag ]
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
Factors affecting solubility
•
•
•
•
Size
Charge
Temperature
Solvent ionic strength
Precipitate
The precipitin reaction
etc.
Zone of equivalence
Antibody/Antigen
Single radial immunodiffusion
Ag
Single radial immunodiffusion
r
r  [Ag ]
Electroimmunodiffusion
• Why would we want to combine
immunodiffusion with electrophoresis?
– SPEED
– Specificity
• Carl-Bertil Laurell (Lund University,
Sweden)
– Laurell Technique (coagulation factors)
– “Rocket electrophoresis”
Electroimmunodiffusion
+
-
Immunoelectrophoresis
• Combines serum protein electrophoresis
with immunometric detection
– Electrophoresis provides separation
– Immunoprecipitation provides detection
• Two related applications:
– Immunoelectrophoresis
– Immunofixation electrophoresis
Immunoelectrophoresis
-human serum
Specimen
+
Immunoelectrophoresis
-
+
P
C

P

C

P

C

Immunofixation electrophoresis
SPE
IgG
IgA
IgM


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
Light reflection
Molecular size and scattering
-
+
-
Distribution of scattered radiation
Nephelometry vs. Turbidimetry
0°-90°
Rate nephelometry
Intensity of scattering
Rate
C1
C2
Time
Additional considerations for
quantitative competitive binding
immunoassays
• Response curve
• Hook effect
%Bound label
Competitive immunoassay
response curve
%Bound vs. log concentration
Antigen concentration
Logistic equation
a
%Bound label
y
c
d
ad
 x
a 
c
Slope = b
Log antigen concentration
b
d
Logit transformation
a
%Bound label
 y 

Y  logit y  ln
 1  y 

y d
where y 
a  d 
d
Log antigen concentration
Logit y
Logit plot
Log antigen concentration
%Bound antigen
High dose “hook” effect
Antigen concentration
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
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
Radioisotope labels
• Advantages
• Disadvantages
– Flexibility
– Sensitivity
– Size
– Toxicity
– Shelf life
– Disposal costs
Enzyme labels
• Advantages
– Diversity
– Amplification
– Versatility
• Disadvantages
– Lability
– Size
– Heterogeneity
Fluorescent labels
• Advantages
– Size
– Specificity
– Sensitivity
• Disadvantages
– Hardware
– Limited selection
– Background
Chemiluminescent labels
• Advantages
– Size
– Sensitivity
– S/N
• Disadvantages
– Hardware
– ?
Chemiluminescent labels
NH 2
O
NH 2
O*
H
N
+
N
H
2 H 2 O 2 + OH -
OO-
Pe r ox i dase
O
+ N2 +
3 H2O
O
L um i n o l
NH 2
COO +
COO -
h
( ma x = 4 3 0 nm )
Chemiluminescent labels
CH 3
Br -
N+
O-
CH 3
N
O
O
+ H 2 O 2 + OH -
O
CO 2 H
A c r i d i n i um e s t e r
+ CO 2 + h
+
CO 2 H
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
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
Enzyme-linked immunosorbent
assay
Substrate
2nd antibody
E
Specimen
S
E
P
E
E
Microtiter well
E
E
ELISA (variation 1)
Specimen
Labeled antigen
E
S
E
P
E
Microtiter well
E
ELISA (variation 2)
Labeled antibody
E
Specimen
E
E
E
E
E
E
Microtiter well
E
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
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
Immobilized antibody methods
• Coated tube
• Coated bead
• Solid phase antibody methods
Coated tube methods
Specimen
Labeled antigen
Wash
Coated bead methods
Microparticle enzyme
immunoassay (MEIA)
Labeled antibody
E
S
P
E
Glass fiber matrix
E
Magnetic separation methods
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Magnetic separation methods
Aspirate/Wash
Fe
Fe
Fe
Fe
Fe
Electrochemiluminescence
immunoassay (Elecsys™ system)
Flow cell
Oxidized
Reduced
Fe
ASCEND (Biosite Triage™)
ASCEND
Wash
ASCEND
Developer
Solid phase light scattering
immunoassay
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
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
Typical design of a homogeneous
immunoassay
No signal
Signal
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
EMIT™ method
S
Enzyme
S
No signal
P
S
Signal
Enzyme
Signal (enzyme activity)
EMIT™ signal/concentration
curve
Functional
concentration range
Antigen concentration
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
Molecular electronic energy
transitions
Singlet
E4
E3
E2
Triplet
VR
E1
IC
A
F
10-6-10-9 sec
P
E0
10-4-10 sec
Polarized radiation
z
x
Polarizing
filter
y
Fluorescence polarization
HO
O
OH
O
C
in
O
Fluorescein
out (10-6-10-9 sec)
Orientation of polarized radiation is maintained!
Fluorescence polarization
in
HO
O
O
C
O
OH
But. . .
out (10-6-10-9 sec)
Rotational frequency  1010 sec-1
Orientation of polarized radiation is NOT maintained!
Fluorescence polarization
immunoassay
Slow rotation
HO
O
OH
Polarization maintained
O
C
HO
O
OH
O
C
O
O
Rapid rotation
Polarization lost
FPIA signal/concentration curve
Signal (I/I)
Functional
concentration range
Antigen concentration
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
Cloned enzyme donor
Donor
Spontaneous
Acceptor
Monomer
(inactive)
Active tetramer
Cloned enzyme donor
immunoassay
Donor
Acceptor
No activity
Donor
Active enzyme
Acceptor
Signal (enzyme activity)
CEDIA™ signal/concentration
curve
Functional
concentration range
Antigen concentration
Other approaches to
homogeneous immunoassay
•
•
•
•
Fluorescence methods
Electrochemical methods
Enzyme methods
Enzyme channeling immunoassay
Substrate-labeled fluorescence
immunoassay
S
Enzyme
S
No signal
Fluorescence
S
Signal
Enzyme
Fluorescence excitation transfer
immunoassay
No signal
Signal
Electrochemical differential
polarographic immunoassay
Oxidized
Reduced
Prosthetic group immunoassay
P
S
Enzyme
No signal
P
P
Signal
Enzyme
Enzyme channeling immunoassay
Substrate
E1
Product 1
E2
Ag
Product 2
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
Molecular imprinting
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)