BÀI GIẢNG CHỦ ĐỀ MIỄN DỊCH

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 funda

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.

• 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.

Aminobenzene Sulphonate, a Hapten

Classification of immunochemical methods

• Particle methods

– Precipitation

• Immunodiffusion

• Immunoelectrophoresis– Light scattering

• Heterogeneous

• Homogeneous

Properties of the antibody-antigen bond

Antibody affinity

Ag Ab

Ag

] ][

[

]

[

Ag Ab

Ag Ab

• Many precipitation methods are qualitative, but

there are quantitative applications, too

Factors affecting solubility

Single radial immunodiffusion

Ag

Single radial immunodiffusion

]

[Ag

r ∝

r

Double immunodiffusion

Örjan OuchterlonyDeveloped double immunodiffusion technique in 1948

Double immunodiffusion (Ouchterlony)

Quantitative double immunodiffusion

• Carl-Bertil Laurell (Lund University, Sweden)

– Laurell Technique (coagulation factors)

– “Rocket electrophoresis”

+ -

• Combines serum protein electrophoresis with

immunometric detection

– Electrophoresis provides separation

– Immunoprecipitation provides detection

• Two related applications:

– Immunoelectrophoresis

– Immunofixation electrophoresis

Specimen

+ -

-Immunofixation electrophoresis

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°

Additional considerations for quantitative

competitive binding immunoassays

• Response curve

• Hook effect

Competitive immunoassay response curve

x a

−

=

1

ln logit

( a d )

d

y y

−

−

=

′ where

High dose “hook” effect

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.

Chemiluminescent labels

+ 2 H2O2 + OH

COO COO -

Chemiluminescent labels

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

– ELISA is the prototype

Enzyme-linked immunosorbent assay

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

Wash

Coated bead methods

Microparticle enzyme immunoassay

Magnetic separation methods

Fe Fe

Fe

Fe

Fe Fe

Fe

Magnetic separation methods

Aspirate/Wash

Electrochemiluminescence immunoassay

(Elecsys™ system)

Flow cell

FeOxidized

Reduced

ASCEND (Biosite Triage™)

Wash

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

• 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™ signal/concentration curve

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

λin

Orientation of polarized radiation is maintained!

λout (10-6-10-9 sec)

Fluorescence polarization immunoassay

O

HO OH

C O O

FPIA signal/concentration curve

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

Cloned enzyme donor immunoassay

CEDIA™ signal/concentration curve

Other approaches to homogeneous

Fluorescence excitation transfer

immunoassay

Signal

No signal

Electrochemical differential polarographic

immunoassay

Oxidized

Reduced

Prosthetic group immunoassay

Enzyme channeling immunoassay

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

Molecular imprinting (Step 1)

N N

Molecular imprinting (Step 2)

N N

Molecular imprinting (Step 3)

N N

Molecular imprinting (Step 4)

Comparison of MIPs and antibodies

Immunoassays using MIPs

• Therapeutic Drugs: Theophylline, Diazepam,

-adrenoceptor antagonist)

• Hormones: Cortisol, Corticosterone

Oligonucleotide-Target complex

Unbound oligonucleotides Aptamer candidates

PCR New oligonucleotide library + Target