Immunochemical Methods and Funtions

Immunochemical Methods in the Clinical Laboratory

Roger L. Bertholf, Ph.D., DABCCChief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville

Associate Professor of Pathology, University of Florida College of Medicine

ASCP/Bertholf

Name The Antigen

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.

• 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.

• 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

• Particle methods

– Precipitation

• Immunodiffusion

• Immunoelectrophoresis

– Light scattering• Nephelometry

• Turbidimetry

• Label methods

– Non-competitive

• One-site

• Two-site

– Competitive

• Heterogeneous

• Homogeneous

• 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

• Non-covalent

• Reversible

• Intermolecular forces

– Coulombic interactions (hydrogen bonds)

– Hydrophobic interactions

– van der Waals (London) forces

• Clonal variation

Antibody affinity

AgAbAgAb

]][[

][

AgAb

AgAbKa

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

• 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

• Size

• Charge

• Temperature

• Solvent ionic strength

Zone of equivalence

The precipitin reaction

Pre

cipi

tate

Antibody/Antigen

etc.

Single radial immunodiffusion

Ag

Single radial immunodiffusion

][Agr r

Double immunodiffusion

Örjan 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”

• 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

• Combines serum protein electrophoresis with immunometric detection

– Electrophoresis provides separation

– Immunoprecipitation provides detection

• Two related applications:

– Immunoelectrophoresis

– Immunofixation electrophoresis

Immunoelectrophoresis

Specimen

-human serum

+

-

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

• 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°

Inte

nsit

y of

sca

tter

ing

Time

Rate nephelometry

Rate

C2

C1

Additional considerations for quantitative competitive binding immunoassays

• Response curve

• Hook effect

• Response curve

• Hook effect

Competitive immunoassay response curve%

Bou

nd la

bel

Antigen concentration

%Bound vs. log concentration

Logistic equation

%B

ound

labe

l

Log antigen concentration

a

d

c

Slope = b

d

cx

a

day b

Logit transformation

%B

ound

labe

l

Log antigen concentration

a

d

y

yyY

1lnlogit

da

dyy

where

Logit plotL

ogit

y

Log antigen concentration

High dose “hook” effect%

Bou

nd a

ntig

en

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

• 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

• 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.

• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.

Radioisotope labels

• Advantages

– Flexibility

– Sensitivity

– Size

• Advantages

– Flexibility

– Sensitivity

– Size

• Disadvantages

– Toxicity

– Shelf life

– Disposal costs

• Disadvantages

– Toxicity

– Shelf life

– Disposal costs

Enzyme labels

• Advantages

– Diversity

– Amplification

– Versatility

• Advantages

– Diversity

– Amplification

– Versatility

• Disadvantages

– Lability

– Size

– Heterogeneity

• Disadvantages

– Lability

– Size

– Heterogeneity

Fluorescent labels

• Advantages

– Size

– Specificity

– Sensitivity

• Advantages

– Size

– Specificity

– Sensitivity

• Disadvantages

– Hardware

– Limited selection

– Background

• Disadvantages

– Hardware

– Limited selection

– Background

Chemiluminescent labels

• Advantages

– Size

– Sensitivity

– S/N

• Advantages

– Size

– Sensitivity

– S/N

• Disadvantages

– Hardware

– ?

• Disadvantages

– Hardware

– ?

Chemiluminescent labels

+ 2H2O2 + OH -

COO -

COO -

O -

O -

+ h ( max = 4 3 0 nm )

+ N2 + 3H2O

NH2

L um i n o l

P e r o x i d a s e

O

O

N

NH

NH2

H

O

O*NH2

Chemiluminescent labels

CH3

N+

CO2H

O O

B r -

Ac r i d i n i um e s t e r

O -

CO2H

+ H2O2 + OH -+ + CO2 + h

O

CH3

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

• 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

• 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

• 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 antibodyE

Substrate

S P

ELISA (variation 1)

Microtiter well

Specimen Labeled antigenE

EEE

S P

ELISA (variation 2)

Microtiter well

Specimen Labeled antibodyE

E E E E

EEE

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

• 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

• Coated bead

• Solid phase antibody methods

Coated tube methods

Specimen Labeled antigen

Wash

Coated bead methods

Microparticle enzyme immunoassay (MEIA)

Labeled antibodyE

E E

S P

Glass fiber matrix

Magnetic separation methods

Fe

Fe

FeFe

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

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

• 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

• 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

• 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 curveS

igna

l (en

zym

e ac

tivi

ty)

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

• 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

E0

E4E3

E2

E1

Singlet

Triplet

A

VR

F

IC

P

10-6-10-9 sec

10-4-10 sec

Polarized radiation

z

y

x

Polarizingfilter

Fluorescence polarization

OHO OH

C

O

O

Fluoresceinin

Orientation of polarized radiation is maintained!

out (10-6-10-9 sec)

Fluorescence polarization

OHO

OH

C

O

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

C

O

O

Polarization maintainedSlow rotation

OHO OH

C

O

O

Rapid rotation

Polarization lost

FPIA signal/concentration curveS

igna

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

• 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 curveS

igna

l (en

zym

e ac

tivi

ty)

Antigen concentration

Functional concentration range

Other approaches to homogeneous immunoassay

• Fluorescence methods

• Electrochemical methods

• Enzyme methods

• Enzyme channeling immunoassay

• Fluorescence methods

• Electrochemical methods

• Enzyme methods

• Enzyme channeling immunoassay

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

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

• 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.

• 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

O

H3C

CH3

N

NO N

NH

O

H3C

CH3

Methacrylic acid+ Porogen

Molecular imprinting (Step 2)

N

NO N

NH

O

H3C

CH3

N

NO N

NH

O

H3C

CH3

Molecular imprinting (Step 3)

N

NO N

NH

O

H3C

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 vivo preparation

• Limited stability

• Variable specificity

• General applicability

• In vitro preparation

• Unlimited stability

• Predictable specificity

• Limited 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

• 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

Aptamer candidates

PCR

New oligonucleotide library

+ Target