Applications of Immunochemical Methods in

the Clinical Laboratory

Roger L. Bertholf, Ph.D.Associate Professor of Pathology

University of Florida College of Medicine

The University of Florida

University of Florida Health Science Center in Gainesville

The University of Florida

University of Florida Health Science Center/Jacksonville

Classification of immunochemical methods

• Particle methods– Precipitation

• Immunodiffusion• Immunoelectrophoresis

– Light scattering• Nephelometry• Turbidimetry

• Label methods– Non-competitive

• One-site• Two-site

– Competitive• Heterogeneous• Homogeneous

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

Types of labels

• Radioactive• Enzyme• Fluorescent• Chemiluminescent

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

The birth of immunoassay• Rosalyn Yalow and

Solomon Berson developed the first radioimmunoassay in 1957

Coated tube methodsSpecimen Labeled antigen

Wash

Coated bead methods

Enzyme-linked immunosorbent assay

Microtiter well

E E E E E

Specimen 2nd antibodyE

Substrate

S P

Microparticle enzyme immunoassay (MEIA)

Labeled antibodyE

E ES P

Glass fiber matrix

Magnetic separation methods

Fe

Fe

Fe Fe

Fe

Fe

FeFe

Fe

Magnetic separation methods

Fe Fe FeFe Fe

Aspirate/Wash

Electrochemiluminescence immunoassay

(Elecsys™ system)

Flow cell

Fe

Oxidized

Reduced

ASCEND (Biosite Triage™)

ASCEND

Wash

ASCEND

Developer

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

SignalEnzyme

S

EMIT™ signal/concentration curve

Sign

al (e

nzym

e ac

tivity

)

Antigen concentration

Functional concentration range

Fluorescence polarization immunoassay (FPIA)

• Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva

• 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

E0

E4E3

E2

E1

Singlet

Triplet

A

VR

F

IC

P10-6-10-9 sec

10-4-10 sec

Polarized radiationz

y

x

Polarizingfilter

Fluorescence polarization

OHO OH

CO

O

Fluoresceinin

Orientation of polarized radiation is maintained!

out (10-6-10-9 sec)

Fluorescence polarization

OHO

OH

CO

O

Rotational frequency 1010 sec-1

in

Orientation of polarized radiation is NOT maintained!

out (10-6-10-9 sec)

But. . .

Fluorescence polarization immunoassay

OHO OH

CO

O

Polarization maintainedSlow rotation

OHO OH

CO

O

Rapid rotationPolarization lost

FPIA signal/concentration curve

Sign

al (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

Substrate-labeled fluorescence immunoassay

Enzyme

S

S Fluorescence

No signal

SignalEnzyme

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

E1

E2

Substrate

Product 1

Product 2

Early theories of antibody formation

• Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces.

• Ehrlich’s 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.

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

O

O-

O

O-

NH 3+

CH2-CH2-CH2-CH3

OH

N

NH2

Cl

Molecular imprinting (Step 1)

N

NO N

NH

OH3C

CH3

N

NO N

NH

O

H3C

CH3

Methacrylic acid+ Porogen

Molecular imprinting (Step 2)

N

NO N

NH

OH3C

CH3

N

NO N

NH

O

H3C

CH3

Molecular imprinting (Step 3)

N

NO N

NH

OH3C

CH3

N

NO N

NH

O

H3C

CH3

Cross-linking monomerInitiating 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 (2-adrenoceptor antagonist)

• Hormones: Cortisol, Corticosterone• Neuropeptides: Leu5-enkephalin• Other: Atrazine, Methyl--glucoside

Aptamers

1014-1015 random sequencesTarget

Oligonucleotide-Target complex

Unbound oligonucleotides

Aptamer candidates

PCR

New oligonucleotide library

+ Target

Thank You!