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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

Name The Antigen

ASCP/Bertholf

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

NH2

NH2

NH2

SO3

SO3
SO3
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

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 [ Ag ]

Double immunodiffusion

rjan Ouchterlony
Developed double immunodiffusion technique in 1948

Double immunodiffusion (Ouchterlony)

Quantitative double immunodiffusion

S3

S4
P

S2

S5
S1

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
-

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

ad
x
a
c

Slope = b

Log antigen concentration

Logit transformation
a

Y logit y ln
1 y

%Bound label

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

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

NH 2

NH 2
N
N

O*

H
+
H

2 H 2 O 2 + OH -

OO-

Pe r ox i dase

+ N2 +

3 H 2O

L um i n o l

NH 2
COO +
COO -

( ma x = 4 3 0 nm )

Chemiluminescent labels

CH 3

Br -

N+
O-

CH 3
N
O

+ H 2 O 2 + OH -

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

Specimen

Substrate

2nd antibody
E

S
E

P
E

Microtiter well

ELISA (variation 1)

Specimen

Labeled antigen
E
S
E

P
E

Microtiter well

ELISA (variation 2)

Labeled antibody
E

Specimen
E
E

E
E

Microtiter well

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)
S

Labeled antibody
E

Glass fiber matrix

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 onesite


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

Enzyme

Signal

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

10-6-10-9 sec

P
E0

10-4-10 sec

Polarized radiation
z

Polarizing
filter

Fluorescence polarization

HO

OH

O
C

in

Fluorescein

out (10-6-10-9 sec)

Orientation of polarized radiation is maintained!

Fluorescence polarization

in

HO

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

OH

O
C

HO

OH

O
C

Polarization maintained

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
Acceptor

Active enzyme

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

Enzyme

Signal

Fluorescence excitation transfer


immunoassay

No signal

Signal

Electrochemical differential polarographic


immunoassay

Oxidized

Reduced

Prosthetic group immunoassay

Enzyme

No signal

P
P

Enzyme

Signal

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

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

Cl
O

NH

3 +

O-

OH

NH2

CH2-CH2-CH2-CH3

Molecular imprinting (Step 1)

Methacrylic acid
+ Porogen

O
H3C
O

NH

N
N

CH3

O
H3C
O

NH

N
N

CH3

Molecular imprinting (Step 2)

O
H3C
O

NH

N
N

CH3

O
H3C
O

NH

N
N

CH3

Molecular imprinting (Step 3)


Cross-linking monomer
Initiating reagent

O
H3C
O

NH

N
N

CH3

O
H3C
O

NH

N
N

CH3

Molecular imprinting (Step 4)

Comparison of MIPs and antibodies


Antibodies

MIPs

In vivo preparation

In vitro preparation

Limited stability

Unlimited stability

Variable specificity

Predictable specificity

General applicability

Limited applicability

Immunoassays using MIPs

Therapeutic Drugs: Theophylline, Diazepam,


Morphine, Propranolol, Yohimbine ( 2-adrenoceptor
antagonist)
Hormones: Cortisol, Corticosterone
Neuropeptides: Leu5-enkephalin
Other: Atrazine, Methyl--glucoside

Aptamers

1014-1015 random sequences

Target

Oligonucleotide-Target complex
Unbound oligonucleotides
+ Target

Aptamer candidates
PCR
New oligonucleotide library