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

Label methods
Non-competitive
One-site Two-site

Light scattering
Nephelometry Turbidimetry

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

The precipitin reaction

Precipitate

etc.

Zone of equivalence Antibody/Antigen

Single radial immunodiffusion

Ag

Single radial immunodiffusion

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
Rate Intensity of scattering C1 C2

Time

Additional considerations for quantitative competitive binding immunoassays

Response curve Hook effect

Competitive immunoassay response curve

%Bound label

%Bound vs. log concentration

Antigen concentration

Logistic equation
a

y=
%Bound label c

ad x a+ c
b

+d

Slope = b

Log antigen concentration

Logit transformation
a %Bound label

y Y = logit y = ln 1 y

( y d) where y = (a d)
d Log antigen concentration

Logit plot

Logit y

Log antigen concentration

High dose hook effect

%Bound antigen

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
NH 1 O N N O L um i n o l H + H 1 1 1 + OH H O Pe r ox i dase O NH 1 O* OO+ N1 +

1 1 H O

NH 1 COO + COO h ( ma x = 111 nm )

Chemiluminescent labels
CH 1 B r N+ CH 1 N O O + H 1 1 + OH O + + CO 1 + h O-

CO 1 H

CO 1 H A c r i d i n i um e s t e r

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 P E E E E

S E

Microtiter well

ELISA (variation 1)
Specimen Labeled antigen E S E E P E

Microtiter well

ELISA (variation 2)
Specimen E E E E E E E Labeled antibody E

Microtiter well

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)

S E P Labeled antibody E 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 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 S Enzyme P Signal No signal

EMIT signal/concentration curve

Signal (enzyme activity)
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 E1 A E0 F

VR

Triplet
IC

10-6-10-9 sec

10-4-10 sec

Polarized radiation
z

Polarizing filter

Fluorescence polarization

HO

OH

O C O

in

Fluorescein

out (10-6-10-9 sec)

Orientation of polarized radiation is maintained!

Fluorescence polarization But. . .

OH O O C O

Rotational frequency 1010 sec-1

Orientation of polarized radiation is NOT maintained!

HO

in

out (10-6-10-9 sec)

Fluorescence polarization immunoassay

Slow rotation
HO O OH O C O

Polarization maintained

HO

OH

Rapid rotation

O C O

Polarization lost

FPIA signal/concentration curve

Functional concentration range

Signal (I/I)

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 Acceptor

Spontaneous

Monomer (inactive) Active tetramer

Cloned enzyme donor immunoassay

Donor Acceptor

No activity

Donor Acceptor

Active enzyme

CEDIA signal/concentration curve

Signal (enzyme activity)
Functional concentration range

Antigen concentration

Other approaches to homogeneous immunoassay

Fluorescence methods Electrochemical methods Enzyme methods Enzyme channeling immunoassay

Substrate-labeled fluorescence immunoassay

S No signal

Enzyme S S Enzyme Fluorescence

Signal

Fluorescence excitation transfer immunoassay

No signal

Signal

Electrochemical differential polarographic immunoassay

Oxidized

Reduced

Prosthetic group immunoassay

Enzyme P

No signal

S P

Enzyme

Signal

Enzyme channeling immunoassay

Substrate E1 E2 Product 1 Product 2

Ag

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)