Download ag-ab react


Lock and Key Concept
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
106
1010
Affinity
Avidity
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

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
Antigen and antibody reactions in the
lab
Precipitation tests
 Agglutination
 ELISA
 Radioimmunoassay
 Immunofluorscence
 Complement Fixation

AGGLUTINATION TESTS
Particulate antigen
• Slide agglutination
• Tube agglutination
• Passive agglutination
+

Slide agglutination test:
Passive Agglutination/Hemagglutination

Agglutination test done with a soluble
antigen coated onto a particle
+

• Applications
– Measurement of Abs to soluble antigens
Coombs (Antiglobulin)Tests
• Incomplete Ab
• Direct Coombs Test
– Detects antibodies on erythrocytes

+
Patient’s RBCs
Coombs Reagent
(Antiglobulin)
Coombs (Antiglobulin)Tests

Indirect Coombs Test
 Detects anti-erythrocyte antibodies in serum
Step 1

+
Patient’s
Serum
Target
RBCs
Step 2
+

Coombs Reagent
(Antiglobulin)
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
Radial Immunodiffusion
• Method
Ab in gel
– Ab in gel
– Ag in a well

Ag
Ag
Ag
Interpretation
proportional to the
concentration

Quantitative
Diameter2
 Diameter of ring is
 Ig levels
Ag Concentration
Ag
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
Countercurrent electrophoresis

Method
 Ag and Ab migrate toward each other by
electrophoresis
 Used only when Ag and Ab have opposite charges
-
+
Ag
• Qualitative
–Rapid
Ab
Complement fixation test
(CFT)

Complement plays an imp role in Ag-Ab reaction.

In the presence of appropriate Abs:
 Complement lyses erythrocytes
 Kills bacteria,
 Immobilizes motile organisms
 Promotes phagocytosis
 Contributes to tissue damage in
hypersensitivity reactions


Principle of CFT: Ability of Ag-Ab complexes to fix
complement

Very sensitive test, can detect minute quantities of
Ag & Ab

All reagents have to be precisely standardised
Eg: Wasserman reaction for sero-diagnosis of
syphilis
Complement Fixation assay
(positive test)
Complement Fixation Assay
(negative test)
Interpretation of result:

Lysis of erythrocytes: Negative CFT

Absence of Erythrocyte lysis: Positive CFT
Indirect CFT: For testing sera that do not fix guinea pig
complement: Hemolysis indicates a positive result
CONGLUTINATING COMPLEMENT ABSORPTION TEST:

Uses horse complement which is non-hemolytic

Indicator system is sheep RBC mixed with bovine serum
which contains a globulin called Conglutinin, which acts
as Ab to the complement
Other complement dependent serological test:
1.
Immune adherence test
2.
Immobilisation test: T.pallidum
3.
Bacterial cytolytic test
NEUTRALIZATION TEST
Virus neutralization tests
2. Bacteriophage neutralization test
3. Toxin neutralization test (exotoxin)
4. Schick test: tests the ability of circulating antitoxin
to neutralize diphtheria toxin injected ID
5. ASO test:
6. Nagler’s reaction: alpha toxin of Cl.perfringens
1.
IMMUNOASSAYS
RIA
 ELISA
 IMMUNOCHROMATOGRAPHY
 CHEMILUMINSCENCE ASSAY
 IMMUNOBLOTTING

Radioimmunoassay
Radioisotopes and enzymes are conjugated to Ag /Ab
 BINDER LIGAND ASSAY


Ag whose concentration is to be determined is called
Ligand (analyte)

RIA is a competitive binding assay in which fixed
amounts of Ab and radiolabelled Ag react in the
presence of unlabelled Ag
Labelled and unlabelled Ags compete for the
limited binding sites on the Ab
Applications of RIA:

Quantitation of hormones. Drugs, tumor
markers, IgE, and viral antigens
Immunoflourescence

Immunofluorescence is an antigen-antibody reaction
where the Abs are tagged (labelled) with a fluorescent
dye and the antigen-antibody complex is
Immunofluorescence
• Direct
– Ab to tissue Ag is labeled with fluorochrome
Fluorochrome
Labeled Ab
Ag
Tissue Section
ELISA (types):
 Direct indirect
 Sandwich
 Competetive
 Casette
BASIC FORMAT
Solid phase = 96 / 384-well microplate
1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen
Analyte = antibody
Analyte = antigen
Incubate, wash
2. Block free binding sites. Incubate. Wash.
Analyte = antibody
Analyte = antigen
3. Add sample. Incubate. Wash
Analyte = antibody
Analyte = antigen
4. Add conjugate. Incubate. Wash.
E
E
Analyte = antibody
E
E
Analyte = antigen
5. Add substrate
6. Incubate, stop, measure colour change
ENZYME
Colourless
OD
CONCENTRATION
AMPLIFICATION
E
Directly conjugated developing
antibody may give weak signal
amplify with
E
E
unlabelled (rabbit) anti-(human) Ig
followed by
anti-(rabbit) Ig-enzyme
or
E
S
E-S B S-E
Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
SUBSTRATES
See Sigma catalogue for list of conjugates and substrates
Orthophenylene diamine
hydrochloride (OPD)
Tetramethyl
benzidine (TMP)
Horse radish peroxidase (HRP)
Orange, 490 nm
Yellow, 450 nm
Spectrophotometer
Paranitrophenyl phosphate (PNP)
Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm
Spectrophotometer
Methyl umbelliferone
365 nm
445 nm
Fluorimeter
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
E
E
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
Useful when pure antigen not available
or antigen coats poorly
1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen
eg tissue homogenate
1. Specific antibody
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash  pure antigen
2. Impure antigen
1. Specific antibody
Types of immunodetection systems
2. Indirect immunodetection
1. Direct immunodetection
Secondary antibody conjugated with
enzyme system
Primary antibody conjugated with
enzyme system
HRP
HRP
HRP
HRP
Ag
Ag
antigen
Ag
Ag
Ag
horseradish
peroxidase
ConsultantHRP
General
Surgeon
3. Sandwich indirect
4. Indirect immunodetection
(A00692Y)
immunodetection
streptavidin
with biotin linkers
Antigen applied in soluble form
Biotinylated primary antibodies
HRP
HRP
HRP
HRP
HRP
HRP
HRP
HRP
HRP
Ag
Ag
Ag
Ag
Streptavidin
Competitive ELISA
Solid Phase Non-Competitive RIA/ELISA

Ab detection
Labeled
Anti-Ig
 Immobilize Ag
 Incubate with sample
 Add labeled anti-Ig
 Amount of labeled Ab
bound is proportional
to amount of Ab in the
sample
• Quantitative
Ab in
Patient’s
sample
Immobilized
Ag
Solid
Phase
Solid Phase Non-Competitive RIA/ELISA

Ag detection
Labeled
Ab
 Immobilize Ab
 Incubate with sample
 Add labeled antibody
 Amount of labeled Ab
bound is proportional to
the amount of Ag in the
sample
• Quantitative
Ag in
Patient’s
sample
Ag
Immobilized
Solid
Phase
Cassette ELISA
Few sample can be
tetsed
Specific antigens are
coated on
nitroclellulose
membrane
These strips are
incubated with
patients serum
positive samples
develop a colred spot
or band
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.
t
HIV-1 Western Blot





Lane1: Positive
Control
Lane 2: Negative
Control
Sample A:
Negative
Sample B:
Indeterminate
Sample C:
Positive
TESTS FOR ANTIGEN-ANTIBODY
REACTIONS
Flow Cytometry
Flow cytometry is commonly used in the clinical laboratory to
identify and enumerate cells bearing a particular antigen. Cells in
suspension are labeled with a fluorescent tag by either direct or
indirect immunofluorescence. The cells are then analyzed on the
flow cytometer.
 The figure at right illustrates the principle of
flow cytometry. In a flow cytometer, the
cells exit a flow cell and are illuminated with
a laser beam. The amount of laser light that
is scattered off the cells as they passes
through the laser can be measured, which
gives information concerning the size of the
cells. In addition, the laser can excite the
fluorochrome on the cells and the
fluorescent light emitted by the cells can be
measured by one or more detectors.

TESTS FOR ANTIGEN-ANTIBODY
REACTIONS


The type of data that is obtained from the flow cytometer is shown
below.
In a one parameter histogram, increasing amount of fluorescence
(e.g. green fluorescence) is plotted on the x axis and the number of
cells exhibiting that amount of fluorescence is plotted on the y axis.
 The fraction of cells
that are fluorescent
can be determined by
integrating the area
under the curve.
 In a two parameter
histogram, the x axis is
one parameter (e.g. red
fluorescence) and the
y axis is the second
parameter (e.g. green
fluorescence). The
number of cells is