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
Name The Antigen
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.
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.


Aminobenzene Sulphonate, a Hapten
NH
2
NH
2
NH
2

SO
3

SO
3

SO
3

Ortho Meta Para
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
Double immunodiffusion
rjan Ouchterlony
Developed double immunodiffusion technique in 1948
Double immunodiffusion (Ouchterlony)
Quantitative double immunodiffusion
S1
S2
S3 S4
S5
P
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
The birth of immunoassay
Rosalyn Yalow (1921-)
and Solomon Berson
described the first
radioimmunoassay in
1957.


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
=430nm)
+N
2
+3H
2
O
NH
2
Luminol
Peroxidase
O
O
N
N
H
NH
2
H
O
O
*
NH
2
Chemiluminescent labels
CH
3
N
+
CO
2
H
OO
Br
-
Acridiniumester
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
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
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.
Fundamentals of antigen/antibody
interaction
CH
2
-CH
2
-CH
2
-CH
3

N
N H
2
C l
Molecular imprinting (Step 1)
N
N O
N
N H
O
H
3
C
CH
3

N
N O
N
N H
O
H
3
C
CH
3

Methacrylic acid
+ Porogen

Molecular imprinting (Step 2)
N
N O
N
N H
O
H
3
C
CH
3

N
N O
N
N H
O
H
3
C
CH
3

Molecular imprinting (Step 3)
N
N O
N
N H
O
H
3
C
CH
3

N
N O
N
N H
O
H
3
C
CH
3

Cross-linking monomer
Initiating reagent
Molecular imprinting (Step 4)
Comparison of MIPs and antibodies
In vivo preparation

Limited stability

Variable specificity

General applicability
In vitro preparation

Unlimited stability

Predictable specificity

Limited applicability
Antibodies MIPs
Immunoassays using MIPs
Therapeutic Drugs: Theophylline, Diazepam,
Morphine, Propranolol, Yohimbine (o
2
-
adrenoceptor antagonist)

Hormones: Cortisol, Corticosterone

Neuropeptides: Leu
5
-enkephalin

Other: Atrazine, Methyl-o-glucoside
Aptamers
10
14
-10
15
random sequences
Target
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target