Principles of Immunology Antigen-Antibody Interactions 4/25/06

Principles of ImmunologyAntigen-Antibody Interactions

4/25/06

Word/Terms List Agglutinin EIA Equivalence zone FIA Immunodiffusion Immunoelectrophoresis RIA Titer

Affinity = attractive and repulsive forces

Ab

Ag

High Affinity

Ab

Ag

Low Affinity

Affinity

• Strength of the reaction between a single antigenic determinant and a single Ab combining site

Specificity The ability of an individual antibody

combining site to react with only one antigenic determinant.

The ability of a population of antibody molecules to react with only one antigen.

Cross Reactivity

• The ability of an individual Ab combining site to react with more than one antigenic determinant.

• The ability of a population of Ab molecules to react with more than one Ag

Anti-A Ab

Ag A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

Factors Affecting Measurement of Ag/Ab Reactions

• Affinity

• Avidity

• Ag:Ab ratio

• Physical form of Ag

Ab excess Ag excess

Equivalence – Lattice formation

Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will

have to depend on lattice formation or they will have to utilize ways to detect small immune complexes

All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Agglutination Tests

Lattice Formation

Agglutination/Hemagglutination

• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin

+

• Qualitative agglutination test– Ag or Ab

Agglutination/Hemagglutination

Quantitative agglutination test Titer Prozone

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

24

Pos.

Neg

.

Titer

648

512<232

128324

Patient

12345678

Agglutination/Hemagglutination

Definition Qualitative test Quantitative test• Applications

– Blood typing– Bacterial infections

–Fourfold rise in titer

• Practical considerations– Easy– Semi-quantitative

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

Passive Agglutination/Hemagglutination

Definition - agglutination test done with a soluble antigen coated onto a particle

+

• Applications– Measurement of antibodies to soluble antigens

Agglutination/Hemagglutination Inhibition

Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

+

Prior to Test

+ +

Test

Patient’s sample

Agglutination/Hemagglutination Inhibition

Applications Measurement of soluble Ag

Practical considerations Same as agglutination test

• Definition

Precipitation Tests

Lattice Formation

Radial Immunodiffusion

Interpretation Diameter of ring

is proportional to the concentration

Quantitative Ig levels

• Method– Ab in gel– Ag in a well

Ag Concentration

Dia

met

er2

AgAgAgAg

Ab in gel

Immunoelectrophoresis

Method Ags are separated by electrophoresis

• Interpretation– Precipitin arc represent individual antigens

Ag-+

Ag

Ab

Ag

Ab

Immunoelectrophoresis

Method Interpretation Qualitative

Relative concentration

Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent

Assays (EIA)

Lattice formation not required

Competitive RIA/ELISA for Ag

Method Determine

amount of Ab needed to bind to a known amount of labeled Ag

+

Prior to Test

Labeled Ag

+

Test

+Patient’ssample

LabeledAg

+– Use predetermined

amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

Solid Phase Non-Competitive RIA/ELISA

Ab detection Immobilize Ag Incubate with

sample Add labeled anti-Ig Amount of labeled

Ab bound is proportional to amount of Ab in the sample

SolidPhase

AgImmobilized

Ab in Patient’s

sample

LabeledAnti-Ig

Solid Phase Non-Competitive RIA/ELISA

Ag detection Immobilize Ab Incubate with sample Add labeled antibody Amount of labeled Ab

bound is proportional to the amount of Ag in the sample

SolidPhase

AgImmobilized

Ag in Patient’s

sample

LabeledAb

Tests for Cell Associated Antigens

Lattice formation not required

Immunofluorescence

• Direct– Ab to tissue Ag is labeled with fluorochrome

Ag

FluorochromeLabeled Ab

Tissue Section

Immunofluorescence

Indirect Ab to tissue Ag is

unlabeled Fluorochrome-labeled

anti-Ig is used to detect binding of the first Ab.

Ag

FluorochromeLabeled Anti-Ig

Tissue Section

UnlabeledAb

• Qualitative to Semi-Quantitative

Assays Based on Complement

Lattice formation not required

Complement Fixation

Ag mixed with test serum to be assayed for Ab– Standard amount of complement is added– Erythrocytes coated with Abs is added– Amount of erythrocyte lysis is determined

Ag

Patient’sserum

Ag No Ag

Ag