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
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