Ag-Ab

reactions:Tests for
Ag-Ab reactions
Dr.Kedar Karki
 Nature of Ag/Ab Reactions
http://www.med.sc.edu:85/chime2/lyso-abfr.htm
• Lock and Key Concept
• 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:

[Ag-Ab]
Keq =
[Ag] x [Ab]
 Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs

Keq = 104 106 1010

Affinity Avidity 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 Anti-A Anti-A

Ab Ab 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/Hemagglutinati
on
 Quantitative agglutination test
 Titer
 Prozone

1/1024
1/256
1/512
1/128
1/16

1/64
1/32

Neg.
Pos.
1/8
1/4
1/2

Patient Titer

1 64
2 8
3 512
4 <2
5 32
6 128
7 32
8 4
 Agglutination/Hemagglutinati
on
 Definition
 Qualitative test

1/256
1/128

1/512
 Quantitative test

1/16

1/64
1/32
1/8
1/4
1/2
• Applications
– Blood typing
– Bacterial infections
–Fourfold rise in titer
• Practical considerations
– Easy
– Semi-quantitative
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

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Coombs (Antiglobulin)Tests
 Indirect Coombs Test
 Detects anti-erythrocyte antibodies in serum

Step 1
+
Patient’s Target
Serum 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

+ +
Patient’s sample
Agglutination/Hemagglutination
Inhibition
 •Applications
Definition
 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 Ag Ag Ag

– Ag in a well
 Interpretation
 Diameter of ring is
proportional to the
concentration Diameter2
 Quantitative
 Ig levels

Ag Concentration
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 Ab

• Qualitative
–Rapid
Radioimmuoassays
(RIA)
Enzyme-Linked
Immunosorbent
Assays (ELISA)
Lattice formation not required
 Competitive RIA/ELISA for Ag
 Method
 Determine amount Prior to Test
of Ab needed to
bind to a known
amount of labeled
+
Ag Labeled
Ag
– Use predetermined
Test
amounts of labeled
Ag and Ab and add a + + +
sample containing Labeled Patient’s
unlabeled Ag as a Ag sample
competitor
 Competitive RIA/ELISA for Ag
 Method cont.
 Determine amount
of labeled Ag Test
bound to Ab
 ↓ NH4SO4 + + +
 ↓ anti-Ig Solid Labeled Patient’s Solid
 Immobilize the Ab Phase Ag sample 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
Labeled
 Immobilize Ag Anti-Ig
 Incubate with sample Ab in
 Add labeled anti-Ig Patient’s
sample
 Amount of labeled Ab
bound is proportional Immobilized Ag
to amount of Ab in
the sample Solid
Phase

• Quantitative
Solid Phase Non-Competitive
RIA/ELISA
 Ag detection
Labeled
 Immobilize Ab
Ab
 Incubate with sample Ag in
 Add labeled antibody Patient’s
sample
 Amount of labeled Ab Ag
bound is proportional to Immobilized
the amount of Ag in the
sample Solid
Phase

• Quantitative
 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
 Fluorochrome-labeled Unlabeled
Labeled Anti-Ig
anti-Ig is used to detect Ab
binding of the first Ab.

Ag
• Qualitative to Semi- Tissue Section
Quantitative
 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

One Parameter Histogram Two Parameter Histogram

Green Fluorescence Intensity

Unstained cells
Number of Cells

FITC-labeled cells

Green Fluorescence Intensity 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

Ag No Ag
Ag
Patient’s
serum
Ag