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
Ag Ab Ag Ab - +
] ][ [
] [
Ag Ab
Ag Ab
K
a
-
=
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
Zone of equivalence
The precipitin reaction
P
r
e
c
i
p
i
t
a
t
e

Antibody/Antigen
etc.
Single radial immunodiffusion
Ag
Single radial immunodiffusion
] [Ag r
r
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
Specimen
o-human serum
+
-
Immunoelectrophoresis
P
C P C P C
k o
+
-
Immunofixation electrophoresis
SPE IgG IgA IgM k
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
I
n
t
e
n
s
i
t
y

o
f

s
c
a
t
t
e
r
i
n
g

Time
Rate nephelometry
Rate
C
2

C
1

Additional considerations for
quantitative competitive binding
immunoassays
Response curve
Hook effect
Competitive immunoassay
response curve
%
B
o
u
n
d

l
a
b
e
l

Antigen concentration
%Bound vs. log concentration
Logistic equation
%
B
o
u
n
d

l
a
b
e
l

Log antigen concentration
a
d
c
Slope = b
d
c
x
a
d a
y
b
+
|
.
|

\
|
+

=
Logit transformation
%
B
o
u
n
d

l
a
b
e
l

Log antigen concentration
a
d
|
|
.
|

\
|
'

'
=
'
=
y
y
y Y
1
ln logit
( )
( ) d a
d y
y

=
'
where
Logit plot
L
o
g
i
t

y

Log antigen concentration
High dose hook effect
%
B
o
u
n
d

a
n
t
i
g
e
n

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
Flexibility
Sensitivity
Size
Disadvantages
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
+ 2H
2
O
2
+ OH
-
COO
-
COO
-
O
-
O
-
+ h (
max
= 4 3 0 nm)
+ N
2
+ 3H
2
O
NH
2
Lumi no l
Pe r o x i d a s e
O
O
N
N
H
NH
2
H
O
O
*
NH
2
Chemiluminescent labels
CH
3
N
+
CO
2
H
O O
B r
-
Ac r i d i n i um e s t e r
O
-
CO
2
H
+ H
2
O
2
+ OH
-
+
+ CO
2
+ h
O
CH
3
N
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
Microtiter well
E E E E E
Specimen
2nd antibody
E
Substrate
S P
ELISA (variation 1)
Microtiter well
Specimen
Labeled antigen
E
E E E
S P
ELISA (variation 2)
Microtiter well
Specimen
Labeled antibody
E
E E E E
E
E
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
E E
S P
Glass fiber matrix
Magnetic separation methods
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Magnetic separation methods
Fe Fe Fe Fe Fe
Aspirate/Wash
Electrochemiluminescence
immunoassay (Elecsys system)
Flow cell
Fe
Oxidized
Reduced
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
Enzyme
S
S P
No signal
Signal
Enzyme
S
EMIT signal/concentration
curve
S
i
g
n
a
l

(
e
n
z
y
m
e

a
c
t
i
v
i
t
y
)

Antigen concentration
Functional
concentration range
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
E
0

E
4

E
3

E
2

E
1

Singlet
Triplet
A
VR
F
IC
P
10
-6
-10
-9
sec

10
-4
-10 sec

Polarized radiation
z
y
x
Polarizing
filter
Fluorescence polarization
O HO OH
C
O
O
Fluorescein

in

Orientation of polarized radiation is maintained!

out

(10
-6
-10
-9
sec)

Fluorescence polarization
O

H
O

O
H

C

O

O

Rotational frequency ~ 10
10
sec
-1

in

Orientation of polarized radiation is NOT maintained!

out

(10
-6
-10
-9
sec)

But. . .
Fluorescence polarization
immunoassay
O HO OH
C
O
O
Polarization maintained
Slow rotation
O HO OH
C
O
O
Rapid rotation
Polarization lost
FPIA signal/concentration curve
S
i
g
n
a
l

(
I
|
|
/
I

)

Antigen concentration
Functional
concentration range
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
Acceptor
Monomer
(inactive)
Active tetramer
Spontaneous
Cloned enzyme donor
immunoassay
Donor
Acceptor
Donor
Acceptor
No activity
Active enzyme
CEDIA signal/concentration
curve
S
i
g
n
a
l

(
e
n
z
y
m
e

a
c
t
i
v
i
t
y
)

Antigen concentration
Functional
concentration range
Other approaches to
homogeneous immunoassay
Fluorescence methods
Electrochemical methods
Enzyme methods
Enzyme channeling immunoassay


Substrate-labeled fluorescence
immunoassay
Enzyme
S
S Fluorescence
No signal
Signal
Enzyme
S
Fluorescence excitation transfer
immunoassay
Signal
No signal
Electrochemical differential
polarographic immunoassay
Oxidized
Reduced
Prosthetic group immunoassay
Enzyme
Enzyme
P
P
S P
Signal
No signal
Enzyme channeling immunoassay
Ag
E
1

E
2

Substrate
Product 1
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)