Ag-Ab reactions

Tests for Ag-Ab reactions

Nature of Ag/Ab Reactions

Lock and Key Concept

http://www.med.sc.edu:85/chime2/lyso-abfr.htm

Non-covalent Bonds
Hydrogen bonds
Electrostatic bonds
Van der Waal forces
Hydrophobic bonds

Multiple Bonds
Reversible
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000

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

Low Affinity

Ab

Ab

Ag

Ag

Affinity = attractive and repulsive forces

Calculation of Affinity

Ag + Ab Ag-Ab
Applying the Law of Mass Action:
Keq =

[Ag-Ab]
[Ag] x [Ab]

Avidity
The overall strength of binding between an Ag
with many determinants and multivalent Abs

Keq =

104
Affinity

106
Avidity

1010
Avidity

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

Cross reactions
Anti-A
Ab

Anti-A
Ab

Anti-A
Ab

Ag A

Ag B

Ag C

Shared epitope

Similar epitope

Factors Affecting Measurement of

Ag/Ab Reactions
Affinity
Avidity

Ab excess

Ag excess

Ag:Ab ratio
Physical form of Ag

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

Neg.

Pos.

1/1024

1/512

1/256

1/128

1/64

1/32

1/16

1/8

1/4

Patient

1/2

Titer
Prozone
Titer

1
2
3

64
8
512

4
5

<2
32

6
7
8

128
32
4

 Bacterial infections
Fourfold rise in titer

Practical considerations
Easy
Semi-quantitative

1/512

1/256

1/128

1/64

1/32

1/16

1/8

1/4

Definition
Qualitative test
Quantitative test
Applications
Blood typing

1/2

Agglutination/Hemagglutination

Passive Agglutination/Hemagglutination
Definition - agglutination test done with a
soluble antigen coated onto a particle

Applications
Measurement of antibodies to soluble antigens

Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes

+
Patients RBCs

Coombs Reagent
(Antiglobulin)

Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
Step 1

+
Patients
Serum

Target
RBCs

Step 2

Coombs Reagent
(Antiglobulin)

Coombs (Antiglobulin)Tests
Applications
Detection of anti-Rh Ab
Autoimmune hemolytic anemia

Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of
agglutination due to competition with a soluble Ag
Prior to Test

Test

Patients sample

Agglutination/Hemagglutination Inhibition
Definition
Applications
Measurement of soluble Ag

Practical considerations
Same as agglutination test

Precipitation Tests
Lattice Formation

Radial Immunodiffusion (Mancini)

Method

Ab in gel

Ab in gel
Ag in a well

Ag

Ag

Ag

Diameter of ring is
proportional to the
concentration

Quantitative

Diameter2

Interpretation

Ig levels
Ag Concentration

Ag

Immunoelectrophoresis
Method
Ags are separated by electrophoresis
Ab is placed in trough cut in the agar

Ag

Ag
Ab

Ag
Ab

Interpretation
Precipitin arc represent individual antigens

Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration

Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by
electrophoresis
Used only when Ag and Ab have opposite charges

+
Ag

Qualitative
Rapid

Ab

Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
Assays (ELISA)
Lattice formation not required

Competitive RIA/ELISA for Ag

Method
Determine amount
of Ab needed to bind
to a known amount
of labeled Ag
Use predetermined
amounts of labeled Ag
and Ab and add a
sample containing
unlabeled Ag as a
competitor

Prior to Test

Labeled
Ag
Test

+
Labeled
Ag

Patients
sample

Competitive RIA/ELISA for Ag

Method cont.
Determine amount
of labeled Ag bound
to Ab
NH4SO4
anti-Ig
Immobilize the Ab

Test

+
Solid Labeled
Ag
Phase

Patients
sample

Solid
Phase

Concentration determined from a standard curve

using known amounts of unlabeled Ag

Quantitative
Most sensitive test

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

Quantitative

Labeled
Anti-Ig
Ab in
Patients
sample
Immobilized

Ag
Solid
Phase

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

Quantitative

Labeled
Ab
Ag in
Patients
sample

Ag

Immobilized
Solid
Phase

Tests for Cell Associated

Antigens
Lattice formation not required

Immunofluorescence
Direct
Ab to tissue Ag is labeled with fluorochrome

Fluorochrome
Labeled Ab

Ag
Tissue Section

Immunofluorescence
Indirect
Ab to tissue Ag is
unlabeled
Fluorochrome-labeled antiIg is used to detect binding
of the first Ab.

Qualitative to SemiQuantitative

Unlabeled
Ab

Fluorochrome
Labeled Anti-Ig

Ag
Tissue Section

Immunofluorescence
Flow Cytometry
Cells in suspension are labeld with fluorescent tag
Direct or Indirect Fluorescence
Cells analyzed on a flow cytometer
Flow
Tip

FL
Detector

Light
Scatter
Detector
Laser

Immunofluorescence
Flow Cytometry cont.
Data displayed
Two Parameter Histogram

Number of Cells

Unstained cells

FITC-labeled cells

Green Fluorescence Intensity

Green Fluorescence Intensity

One Parameter Histogram

Red Fluorescence Intensity

Assays Based on Complement

Lattice formation not required

Complement Fixation
Methodology

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

No Ag

Ag
Ag
Ag

Patients
serum