Ag-Ab reactions

Tests for Ag-Ab reactions

 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/Hemagglutination
• 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/Hemagglutination
• Definition
• Qualitative test

1/256
1/128

1/512
1/16

1/64
1/32
1/8
1/4
1/2
• Quantitative test
• 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

• 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 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 of Prior to Test
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 bound Test
to Ab
+ +  +
•  NH4SO4 Solid Labeled Patient’s Solid
•  anti-Ig Phase Ag sample Phase
• Immobilize the Ab
– 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
Ab in
– Incubate with sample Patient’s
– Add labeled anti-Ig sample

– Amount of labeled Ab Immobilized Ag

bound is proportional
Solid
to amount of Ab in the Phase
sample
• Quantitative
 Solid Phase Non-Competitive RIA/ELISA

• Ag detection
Labeled
– Immobilize Ab Ab
– Incubate with sample Ag in
Patient’s
– Add labeled antibody sample Ag
– Amount of labeled Ab Immobilized
bound is proportional to
the amount of Ag in the Solid
Phase
sample
• 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
Labeled Anti-Ig
– Fluorochrome-labeled anti- Unlabeled
Ab
Ig is used to detect 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