Antigen-Antibody interactions
Principles & Applications
Dr. M. Izad
Oct 2009
the nature of Ag-Ab reactions
 Reversible
 Specificity
 Non-covalent
 Lock
Bonds
and Key Concept
Non-covalent interactions
form the basis of Ag-Ab binding
Figure 3-7 part 2 of 2
Complementarity
determining region
Epitope & Paratope
Affinity & Avidity

The combined strength of the
noncovalent interactions between
a single Ag-binding site and a
single epitope is the affinity of the
antibody

Antibody affinity is a quantitative
measure of binding strength

The
strength
of
multiple
interactions between a multivalent
antibody and antigen is called
avidity
Measuring affinity of Ab to Ag
Ag + Ab ⇆ Ag-Ab
[Ag - Ab]
Ka 
[Ag] [Ab]
Low affinity Ag-Ab complex: ka=104-105 L/mol
High affinity Ag-Ab complex: ka=1011 L/mol
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq =
104
Affinity
106
Avidity
1010
Avidity
Cross-reactivity
 In
some cases, antibody elicited by one antigen
can “cross-react” with an unrelated Ag
 Cross-reactivity occurs if two different antigens
share an identical/similar epitope)
Cross reactions
Anti-A
Ab
Anti-A
Ab
Anti-A
Ab
Ag A
Ag B
Ag C
Identical epitope
Similar epitope
Cross-reactivity
Examples:
-ABO Blood groups antigens
-Bacteria
&
Viruses
pyogenes, Vaccinia virus)
(Streptococcus
Polyclonal
Antibodies
Monoclonal
Only the targeted tumor cells are killed
by the immunotoxin
Bunch of other toxins
could be used
Ag-Ab intractions Invitro
•Percipitation
•Agglutination
•Hemagglutination
•Passive Agglutination
•Agglutination Inhibition
•Modern Tests
Precipitation is a Classical Demonstration of
Antibody-Antigen Interaction
Precipitation Reactions in Gels
(Mancini Method)
(Ouchterlony
Method)
Radial Immunodiffusion (Mancini)
Ab in gel
Ag
Ag
Diameter2
Ag
Ag Concentration
Ag
Nephelometry
Immunoelectrophoresis
Agglutination /Hemagglutination
Reaction
 Interaction
between antibody and a particulate
antigen
 Antibodies
that produce such reactions are
called agglutinin
 Excess
of antibody can also inhibit agglutination
reactions (prozone effect)
 Some
antibodies bind but do not agglutinate
(Incomplete antibodies)
Agglutination Tests

Blood typing

Diagnosis of infectious disease

Typing of bacteria

Passive agglutination is useful with Soluble
antigens
Quantitative agglutination test
Neg.
Pos.
1/1024
1/512
1/256
1/64
1/32
1/16
1/8
1/4
1/2
Patient
1/128

+
Titer
1
64
2
8
3
512
4
<2
5
32
6
128
7
32
8
Prozone
4
Agglutination inhibition assays
Radioimmunoassay (RIA)
used for measuring hormones, serum proteins,
drugs, etc. at low concentration (≤ 0.001ug/ml)
Enzyme-linked Immunosorbent Assay
(ELISA)
Types of ELISA
Western Blot

Identification of a
specific protein in a
complex mixture of
proteins

Identification of a
specific antibody in a
mixture
Immunoprecipitation
Immunofluorescence
Three commonly used fluorescent
dyes
(fluorochrome)
that
are
covalently bound to antibodies:
Fluorescein (490 → 517)
Rhodamine (515 →546)
Phycoerythrin
Fluorescence
Microscope
Fluorescein
-excited by blue
light (450-490)
-emits green
light (520-560)
Fluorescent anti-IgG staining of kidney biopsy
from a patient with Goodpasture’s syndrome
Flow cytometry