Title: Antigen
1Antigen Antibody Reactionsor Serological
Reactions
2Antigen-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
3Introduction
- 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
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5Strength 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
6Strength 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
)
7CROSS 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
8STAGES 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
9LATTICE 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)
10Zone of equivalence
11LATTICE 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
12ZONES OF PRECIPITIN FORMATION
Precipitin Curve
13METHODS 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
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15Precipitation
- Precipitation can take place in capillary tubes,
test tubes, and in gel
16Precipitation in gel
- - Double diffusion
-
- - Single (radial) diffusion
-
- - Combination of diffusion in gel and
- electrophoresis
17SINGLE 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
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19RADIAL 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
3-standard curve
20RADIAL IMMUNODIFFUSION
Samples
Standards
Precipitin Rings
a b c
A B C
Standard Curve
21RADIAL 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
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23OUCHTERLONY 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,
non-identity, partial identity
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25OUCHTERLONY DOUBLE DIFFUSION
Ouchterlony Plates Precipitin
Patterns
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28ELECTROIMMUNOASSAY (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
29ELECTROIMMUNOASSAY (ROCKET)
30ELECTROIMMUNOASSAY (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
31IMMUNONEPHELOMETRY
- 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
32IMMUNOELECTROPHORESIS (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
3333
Immunoelectrophoresis of normal human serum.
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36IMMUNOELECTROPHORESIS (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
37IMMUNOFIXATION 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
38IFE
39IMMUNOFIXATION 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
40WESTERN 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
41WESTERN BLOTTING
42The Western blot procedure.
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43FLOCCULATION
- 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)
44FLOCCULATION VS. IMMUNOPRECIPITATION
45- Flocculation Tests
- VDRL (Venereal Disease Research Lab.) test
- RPR (Rapid Plasma Reagin) test
46Agglutination
- Titer
- Zeta potential
- Types of Agglutination
- - Direct agglutination or hemagglutination
- - Indirect (passive) agglutination or
hemagglutination - - Agglutination or hemagglutination inhibition
- The Coombs test
- - Direct
- - Indirect
47Agglutination Reactions
48Agglutination
- Qualitative slide agglutination
- - identification of bacteria with antisera
directed against - O, H, K antigens
49Agglutination
- Latex agglutination
- Coagglutination
50Agglutination
- Tube agglutination tests
- - Gruber-Widal typhoid fever (S. typhi)
- - Weil-Felix typhus (Rickettsia)
- - Wright brucellosis
- Identify and titrate antibodies in the patients
- serum.
- Titre is defined as the reciprocal of the
- highest dilution of serum showing agglutination.
511200
1400
1100
Titer
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53Agglutination inhibition
54Hemagglutination 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
55Use of Labels in Ag Ab Reactions
- Immunoassays
- - Radioimmunoassay (RIA)
- - Enzyme Immunoassys (EIA)
- Immunofluorescence (IF)
- - Direct IF
- - Indirect IF
- Flow cytometry and Cell Sorting (FACS)
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58Immunologic 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
59ELISA
60ELISA tests
- Depend on enzyme conjugated to 2 Ab reacting with
a specific substrate to produce a color reaction.
- Variations of ELISAs 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
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62Direct and indirect Immunofluorescence
63Immunoprecipitation
- Provides a quick and sensitive test for finding
proteins/Ags especially in low concentrations - Binds Ab to synthetic bead support ? centrifuged
- Or 2 Ab with bead or magnetic bead and collect
by magnetism
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65Distribution of selected markers on some leukemia
cell types ? Immunophenotyping using flow
cytometry mAb
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