Antigen – Antibody Reactions
or
Serological Reactions
Antigen-Antibody interactions
Characterized as:
Non-covalent interaction (similar to “lock and
key” fit of enzyme-substrate)
Do not lead to irreversible alteration of Ag or Ab
This exact and specific interaction has led to
many immunological assays that are used to:
detect Ag or Ab
diagnose disease
measure magnitude of humoral IR
identify molecules of biological and
medical interest
Introduction
Ag – Ab reactions are one of the most specific
noncovalent biochemical reactions known
The forces that hold the reactants together are:
- van der Waals forces
- Electrostatic forces
- Hydrophobic forces
They can be represented by the simple formula:
Ag + Ab ↔ AgAb
The reaction is driven to the right but it is reversible
Strength of Reaction
The strength of the reaction (how far it is driven
to the right) is referred to as affinity
Antibody affinity
- A quantitative measure of binding strength
- Combined strength of the noncovalent
interactions between a binding site on an Ab &
monovalent Ag
- Affinity varies broadly among immunoglobulins
Strength of Reaction
Antibody avidity
- Avidity is often used to describe the collective affinity
of multiple binding sites on an antibody molecule
- True strength of the Ab -Ag interaction within
biological systems
- The interaction at one site will increase the possibility
of reaction at a second site
- High avidity can compensate for low affinity (secreted
pentameric IgM has a higher avidity than IgG )
CROSS REACTIVITY
Antibody elicited by one Ag can cross-react with
a related Ag.
Occurs if two different Ags share identical or very
similar epitope
1- Vaccinia virus and smallpox virus
2- Rabies & JE vaccine
3- Streptococcus pyogenes infection: heart &
Kidney damage following infection
4- Original antigenic sin.
5- Bacterial Ag and sugars on RBC
STAGES OF Ag
- Ab REACTIONS
Primary reactions Vs secondary reactions: Small Ag Ab complexes Vs large complexes (The Lattice
hypothesis)
Development of macroscopic manifestations reactions
(e.g. immunoprecipitation)
Ag – Ab reactions involving IgM are confined to the
blood stream, while those of lower molecular weight
(IgG and IgE) can leave the vasculature and enter
tissues
Time required is hours to days for precipitin formation
leading to irreversible immunoprecipitates
LATTICE THEORY
Lattice formation (visible Ag - Ab aggregates)
occurs when:
– Ag is multivalent (contains more than 2
identical epitopes)
– Cross-linking of Ags by specific Abs (2 or
more antigen-binding sites)
– Molar ratios of epitopes and antigen-binding
sites are optimal (zone of equivalence)
Zone of
equivalence
LATTICE THEORY
Zones of lattice formation
– Far Ag excess (no ppt. formed; free Ag in
supernatant) -- “postzone”
– Ag excess (sub-optimal ppt.; free Ag in spnt.)
– Zone of equivalence (maximum ppt.; no Ag or Ab in
spnt.)
– Ab excess (sub-optimal ppt; Ab in spnt.)
– Far Ab excess (no ppt; Ab in spnt.) -- “prozone”
ZONES OF PRECIPITIN FORMATION
Precipitin Curve
METHODS THAT DETECT Ag- Ab REACTIONS
Primary Reactions:
- Immunofluorescence (IF)
- Radioimmunoassay (RIA)
- Enzyme immunoassay (EIA)
- Immunonephelometry (measures picogram to
nanogram quantities of analyte)
Secondary Reactions
- Agglutination Techniques
- Precipitation Techniques ± Electrophoresis
Precipitation
Precipitation can take place in capillary tubes,
test tubes, and in gel
Precipitation in gel
- Double diffusion
- Single (radial) diffusion
- Combination of diffusion in gel and
electrophoresis
SINGLE VS. DOUBLE DIFFUSION
Single diffusion
– Supporting medium (gel) contains one
reactant at a uniform concentration
– Only the unknowns move through the
medium
Double diffusion
– Gel is inert (contains no reactants)
– Both Ag and Ab travel through the medium
The region of
equivalence
RADIAL IMMUNODIFFUSION
Ab uniformly distributed in gel; Ag diffuses outward
from a well (single diffusion)
Ag- Ab complexes form as concentric rings around the
well at zone of equivalence
At a set time, ring diameters are measured
[Ag] is directly proportional to the ring d2
Unknown value is determined by comparing to a 3standard curve
RADIAL IMMUNODIFFUSION
Standards
Precipitin Rings
Standard Curve
A
B C
Samples
a b
c
RADIAL IMMUNODIFFUSION
Fahey method (kinetic)
– Read at 18 hours
– Plot [std] vs. ring diameter on semi-log paper
Mancini method (endpoint)
– Read at 48 or 72 hours
– Plot [std] vs. ring diameter squared on graph paper
Results reliable only if the ring size is within the range
of the standards; if greater than highest std, dilute and
repeat test
Used to measure IgM, IgG, C4,C3,transferrin, CRP,
others
OUCHTERLONY DOUBLE DIFFUSION
Ag & Ab placed in wells cut into an agarose gel
(both reactants diffuse)
Precipitin line (or arc) indicates Ab has
specificity for Ag
Position of precipitin between wells depends on
MW and concentration of reactants
3 possible patterns of reaction: identity, nonidentity, partial identity
OUCHTERLONY DOUBLE DIFFUSION
Ouchterlony Plates
Precipitin Patterns
ELECTROIMMUNOASSAY (ROCKET)
Electrophoresis hastens movement of Ag (placed in
wells) through Ab -imbedded gel (single diffusion)
Selected pH (8.6) keeps Abs at their isoelectric point;
they will not move
Rocket-shaped precipitin bands will form at zone of
equivalence (changes as reactants move)
[Ag] proportional to length of rocket
Unknowns compared to standards
ELECTROIMMUNOASSAY (ROCKET)
ELECTROIMMUNOASSAY (ROCKET)
May be used to quantitate plasma proteins such
as coagulation factors, alpha-fetoprotein, C3,
C4, CRP, haptoglobin
Compared with RID:
– faster
– similar sensitivity
– requires electrophoretic equipment and more
technological finesse
Largely replaced by immunonephelometry
IMMUNONEPHELOMETRY
Ag + Ab  AgAb  microscopic Ag - Ab complexes
Microcomplexes cause light moving through the
suspending solution to scatter
Nephelometer detects light scattered at a 90o angle
Amount of light scattered at 90o is proportional to Ag Ab complexes formed
Sensitive and quantitative technique used for
measurement of many serum proteins
IMMUNOELECTROPHORESIS (IEP)
Electrophoresis and double diffusion
2 stages
– Proteins separated by electrophoresis
– Antiserum placed in trough parallel to separated
proteins; all reactants diffuse in all directions
– Precipitin forms at zones of equivalence
Trough may be filled with simple or complex antisera
yielding simple to complex patterns
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Immunoelectrophoresis of normal human serum.
IMMUNOELECTROPHORESIS (IEP)
Qualitative to semi-quantitative
Serum, urine, or CSF may be analyzed
Complex patterns may be difficult to interpret
Useful to detect:
– missing proteins
– abnormal proteins
– normal proteins in abnormal concentrations
Used to evaluate conditions such as multiple myeloma
Largely replaced by immunofixation
IMMUNOFIXATION
ELECTROPHORESIS (IFE)
Proteins that were separated by electrophoresis are
exposed to Ab directly, instead of through diffusion
Steps:
– Electrophoresis of protein mixture in gel (use
serum or urine samples)
– Paper strips imbedded with specific Ab are “blotted”
onto gel; Ags transfer to paper and bind to Abs
– Strips washed (unbound material washes away)
– Strips stained to reveal precipitin bands
IFE
IMMUNOFIXATION
ELECTROPHORESIS (IFE)
Used to detect the presence of Igs in conditions
like multiple myeloma
Fairly sensitive - Ab is highly specific,
electrophoresis leaves Ag isolated and
accessible
Faster and easier to interpret than IEP
Only 1 Ab may be used per strip
WESTERN BLOTTING
Similar to IFE but the unknown is Ab rather than Ag
Steps:
– Separation of complex antigenic material (eg., viral
proteins) by electrophoresis
– Separated components transferred from gel to
nitrocellulose paper by “blotting”
– Unknown (or control) sera (which may have Abs)
incubated with paper strips; Ag - Ab complexes ppt.
at site of transfer
– Strips washed; staining reveals complexes
WESTERN BLOTTING
The Western blot procedure.
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FLOCCULATION
Immunoprecipitation (or agglutination) of
insoluble particles
Characterized by very sharp pro- and
postzones
No precipitin formed in zones of Ab or Ag
excess, only in zone of equivalence
Clinically important examples, VDRL and RPR
tests (screening tests for syphilis)
FLOCCULATION VS. IMMUNOPRECIPITATION
Flocculation Tests
- VDRL (Venereal Disease Research Lab.) test
- RPR (Rapid Plasma Reagin) test
Agglutination
Titer
Zeta potential
Types of Agglutination
- Direct agglutination or hemagglutination
- Indirect (passive) agglutination or hemagglutination
- Agglutination or hemagglutination inhibition
The Coombs test
- Direct
- Indirect
Agglutination Reactions
Agglutination
• Qualitative slide agglutination
- identification of bacteria with antisera directed against
O, H, K antigens
Agglutination
• Latex agglutination
• Coagglutination
Agglutination
• Tube agglutination tests:
- Gruber-Widal: typhoid fever (S. typhi)
- Weil-Felix: typhus (Rickettsia)
- Wright: brucellosis
Identify and titrate antibodies in the patient’s
serum.
Titre: is defined as the reciprocal of the
highest dilution of serum showing agglutination.
1:100
1:200
Titer
1:400
Agglutination inhibition
Hemagglutination Inhibition Test
To Detect Antibodies (Rubella)
- Serum (Ab)+ HA +RBCs= No Hemagglutination
= Positive Test
- Serum (No Ab)+ HA + RBCs =Hemagglutination
=Negative Test
To Detect Antigen (HBsAg)
- Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs =
No Hemagglutination = Positive Test
- Serum (No HBsAg)+ Anti HBsAG + HBsAg coated
RBCs = Hemagglutination =Negative Test
Use of Labels in Ag – Ab Reactions
Immunoassays
- Radioimmunoassay (RIA)
- Enzyme Immunoassys (EIA)
Immunofluorescence (IF)
- Direct IF
- Indirect IF
Flow cytometry and Cell Sorting (FACS)
Immunologic Tests
4) Radioimmunoassay (RIA)– a very sensitive test;
used for measuring hormones, serum proteins, drugs,
etc. at low concentrations (≤ 0.001ug/ml)
measures “competitive binding” of radiolabelled Ag
+ unlabelled (test) Ag to high affinity Ab
ELISA
ELISA tests
Depend on enzyme conjugated to 2 Ab reacting with a
specific substrate to produce a color reaction.
Variations of ELISA’s: Allows for qualitative or
quantitative testing. Each one can be used for
qualitative detection of Ag or Ab
Also, a standard curve based on known
concentrations of Ag/Ab can be prepared and an
unknown concentration can be determined
Indirect ELISA
Sandwich ELISA
Competitive ELISA
Direct and indirect
Immunofluorescence
Immunoprecipitation
Provides a quick and
sensitive test for finding
proteins/Ag’s especially
in low concentrations
Binds Ab to synthetic
bead support 
centrifuged
Or 2° Ab with bead or
magnetic bead and
collect by magnetism
Distribution of selected markers on some leukemia
cell types → Immunophenotyping using
“flow cytometry & mAb”
Sensitivity of various immunoassays