Antigen and antibody reactions
• Antigen reacts with a specific antibody in
an observable manner
• Ag-Ab reaction in vitro - serology
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
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq =
104
Affinity
106
Avidity
1010
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
Ab
Anti-A
Ab
Anti-A
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
Antigen antibody tests
• Used in both directions
• Qualitative
• Quantitative
Antigen and antibody reactions in
the lab
•
•
•
•
•
•
Precipitation tests
Agglutination
ELISA
Radioimmunoassay
Immunofluorscence
Complement Fixation
Qualitative/quantitative
• Qualitative
– determines antigen or antibody is present or
absent
• Quantitative
– determines the quantity of the antibody
– Titer
– The highest dilution of the specimen usually
serum which gives a positive reaction in the
test
Precipitation tests
• Soluble antigen reacts with specific
antibody in presence of electrolytes at an
optimal temp and pH to form an insoluble
precipitate
• Flocculation – precipitate remains
suspended as floccules
Prozone phenomenon
• Precipitation takes place when there is
optimal proportions of Antigen and
antibodies
• Absence of precipitation in presence of
excess of antibodies
• Such serum should be diluted before doing
precipitation tests
ZONE of
Equivalence
No Soluble Ag or Ab
ANTIGEN
EXCESS
Ab CONC
ANTIBODY
EXCESS
Mechanism of precipitation
• Marrack’s lattice
hypothesis
• Precipitation occurs
whenever multivalent
antigens combine with
bivalent antibodies to
from a large lattice
• Same hypothesis
applies for
agglutination also
Equivalence – Lattice formation
Precipitation tests
• Precipitation techniques
• Tube precipitation test -Ring test – Ascoli’s
thermo precipitation test
• Flocculation
• Slide – VDRL for syphilis
• Tube – Khan test for syphilis
Ring test
VDRL
Immunodiffusion test
• Precipitation in 1% agar gel
• Single diffusion in one dimension – Oudin
• Double diffusion in one dimension –
Oklaey-Fulthrope
• Single diffusion in two dimensions - Radial
Immunodiffusion
Radial Immunodiffusion (Mancini)
• Method
Ab in gel
– Ab in gel
– Ag in a well
Ag
Ag
Ag
– Diameter of ring is
proportional to the
concentration
• Quantitative
– To estimate
Immunoglobulin
levels
Diameter2
• Interpretation
Ag Concentration
Ag
In this example, Anti-dog IgG is
Mixed in agar so only what is
Placed in wells (Ag) diffuses out
Double diffusion in two dimensions
– Ouchterlony – Elek’s test
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; Myeloma proteins
22
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
•Hepatitis B Ag, Cryptococcus antigen in CSF
Rocket electrophoresis
Applications of precipitation
reactions
• Identification of microbial antigens Anthrax bacilli, Streptococcus,
Cryptococcus
• Blood grouping
• VDRL
• Forensic applications
• Estimation & Identification of
Immunoglobulins
NEUTRALIZATION TESTS
• Detection of bacterial toxins by using
antibodies
– Toxigenicity tests – detection of toxins
produced by C.diptheriae, Cl.welchi etc
– Antistreptolysin ‘O’ test
• Viral neutralisation test
Neutralization tests
• To measure virus neutralizing antibodie in patient’s
serum
• Serum( in dilutions) + Viral suspension inoculated
into test cell culture
• Viral suspension inoculated into control cell culture
• Failure to develop CPE if Antibodies present
• CPE in control cell culture
• Four fold rise in Ab titer is required to establish
diagnosis
IMMUNOASSAYS
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•
•
•
•
RIA
ELISA
IMMUNOCHROMATOGRAPHY
CHEMILUMINSCENCE ASSAY
IMMUNOBLOTTING
RadioImmunoassay
• The radioactivity of the specific labeled
antibody or antigen is used to quantify the
antigen or antibody in patient’s serum
• Radioimmunoassay
s (RIAs) utilize a
radioactive label
(usually 125I, 3H
or 14C), which
emits radiation that
can be measured
with a beta or
gamma counter.
Solid Phase RIA for antigen/direct
• Ag detection
–
–
–
–
Immobilize Ab
Incubate with sample
Add labeled antibody
Amount of labeled Ab
bound is proportional to
the amount of Ag in the
sample
• Quantitative
Labeled
Ab
Ag in
Patient’s
sample
Ag
Immobilized
Solid
Phase
Solid Phase RIA for antibody/indirect
• Ab detection
–
–
–
–
Immobilize Ag
Incubate with sample
Add labeled anti-Ig
Amount of labeled Ab
bound is proportional
to amount of Ab in the
sample
• Quantitative
Labeled
Anti-Ig
Ab in
Patient’s
sample
Immobilized
Ag
Solid
Phase
ELISA
technique
biochemical technique used
Is a
mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
• The technique is divided into
1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
3- Indirect ELISA
Competitive ELISA
• The labelled antigen competes for primary
antibody binding sites with the sample antigen
(unlabeled).
• The more antigen in the sample, the less
labelled antigen is retained in the well and the
weaker the signal).
Sandwich ELISA
• The ELISA plate is
coated with
Antibody to detect
specific antigen
Indirect ELISA
Cassette ELISA
Few sample can be
tetsed
Specific antigens are
coated on
nitroclellulose
membrane
These strips are
incubated with
patients serum
positive samples
Enzyme Immunoassay
• Enzymes used include:
–
–
–
–
Horseradish peroxidase
Glucose-6-phosphate dehydrogenase
Alkaline phosphatase
Β-D-galactosidase
Immunochromatography
•
Chemiluminescent
Immunoassays
The process of chemiluminescence occurs when energy in the
form of light is released from matter during a chemical
reaction.
Chemiluminescent
Immunoassays
• Can be used for heterogeneous or
homogeneous assays.
• Can attach label to antigen or antibody.
• Heterogeneous assays use competitive and
sandwich assay.
• Competitive assays used to measure smaller
analytes.
• Sandwich assays are used to measure larger
analytes.
Chemiluminescent Immunoassay
•
•
•
•
Many applications.
Can measure antigen or antibody.
Add chemiluminescently tagged analyte.
Measure light which is emitted which is directly related to
concentration although competitive binding assays are available.
Immunoblotting
Western Blot
HIV-1 Western Blot
• Lane1: Positive
Control
• Lane 2: Negative
Control
• Sample A: Negative
• Sample B:
Indeterminate
• Sample C: Positive