Beruflich Dokumente
Kultur Dokumente
Diagnostic Immunology
Professor Md. Akram Hossain
MMC
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6.
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Agglutination
Precipitation
Complement fixation
Neutralization
Antibody dependant cell mediated
cytotoxicity (ADCC)
Immobilization
Prof. Md. Akram, MMC
7.
8.
Agglutination
Precipitation
Neutralization
Complement fixation
Fluorescent--antibody technique
Fluorescent
ELISA-- Enzyme linked immunosorbent
ELISA
assay
Radio immunoassay
ImmunochromatographY (ICT)
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Autoimmune disease
By detecting antibodies against particular self antigen in
case of autoimmune diseases
Tumors
By detecting tumor markers.
markers.
Metabolic diseases
Physiological conditions
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Autoimmune diseases
Tumors
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similar
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Agglutination
The term agglutination came from glu
glu-which means adhesion.
adhesion.
The act of adhesion of different parts is
agglutination..
agglutination
When an antibody reacts with a
multivalent
particulate
(insoluble)
antigen,, lattice formation occurs due to
antigen
cross linking of various antigen particles
by the antibody
antibody..
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Types of Agglutination
Direct agglutination
1. Slide Blood grouping, Serotyping of bacteria
2. Tube Widal test (Classical)
Hemagglutination
Latex agglutination
Particle agglutination
Co--agglutination
Co
Flocculation tests
Coombs test
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Disadvantages
Not highly specific
Not highly sensitive
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Direct agglutination
Occurs when the
antigenic
determinant
is
inherent
to
the
particle
itself..
itself
(naturally)
Example #1 Using
group A rbcs to
detect
antianti-A
in
serum..
serum
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Direct agglutination..2
Example # 2 Using
bacteria (Ag) looking
for Ab in serum.
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Passive
Agglutination/Hemagglutination
Definition - agglutination test done
with a soluble antigen coated onto
a particle
Applications
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Latex agglutination
In latex agglutination
procedures,
Ag
molecules
can
be
bound to the surface of
latex beads
beads..
If Ab is present in the
test specimen, the Ag
will combine with the
Ab and form visible
aggregates..
aggregates
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Latex agglutination
In latex agglutination
procedures,
Ag
molecules
can
be
bound to the surface of
latex beads
beads..
If Ab is present in the
test specimen, the Ag
will combine with the
Ab and form visible
aggregates..
aggregates
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Latex agglutination
Latex particles can be
coated with Ab, and in
the presence of Ag
can
form
visible
aggregates..
aggregates
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Hemagglutination
Agglutination of rbcs
as a result of Ab
interaction
with
antigenic determinants
on rbcs surfaces
surfaces..
Example
using
group A rbcs to detect
anti--A in serum.
anti
serum.
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Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes
+
Patients RBCs
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Coombs Reagent
(Antiglobulin)
Prof. Md. Akram, MMC
22
Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti
anti--erythrocyte antibodies in
serum
Step 1
+
Patients
Serum
Target
RBCs
Step 2
+
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Coombs Reagent
Prof. Md. Akram, MMC
(Antiglobulin)
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Coombs (Antiglobulin)Tests
Applications
Detection of anti
anti--Rh Ab
Autoimmune hemolytic anemia
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Flocculation tests
Flocculation tests for Ab detection
are based on the interaction of
soluble Ag with Ab, which results
in the formation of a precipitate of
fine particles
particles.. (Ag consists of lipid
type particles)
Examples VDRL & RPRs.
RPRs.
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Precipitation
Precipitation : Means a deposit on
the earth of hail, mist, rain, sleet, or
snow;; also, the quantity of water
snow
deposited..
deposited
When
soluble
antigens
and
antibodies are mixed together at
lattice
optimum
concentration,
formation occurs
occurs..
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Types of precipitation
1.
Precipitation in gel
Single radial immunodiffusion
Double diffusion
2.
Precipitation in Electrophoresis
Immune electrophoresis
Counter current Immune
electrophoresis (CIE)
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Disadvantages
Time consuming
Some costly instruments are required
High technical skill required
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Ab in gel
Ab in gel
Ag in a well
Ag
Ag
Ag
Ag
Diameter of ring
is proportional
to the
concentration
Quantitative
Diameter2
Interpretation
Ig levels
Ag Concentration
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Immunoelectrophoresis
Method
Ags are separated by electrophoresis
Ab is placed in trough cut in the agar
Ag
Ag
Ab
Ag
Ab
Interpretation
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Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration
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Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by
electrophoresis
Used only when Ag and Ab have opposite
charges
+
Ab
Ag
Qualitative
Rapid
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CFT
Principle: Antigen
Principle:
Antigen-- antibody (IgG, IgM)
complex activates the complement which
can lyse target (RBC).
(RBC).
Components of test:
test:
1. Sensitised sheep RBC (Sheep RBC+ Anti
sheep RBC)
2. Complement
Complement-- ( Guniea pig serum)
3. Known Ag / known Ab
Movie
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titer
may
be
too
low
for
agglutination/precipitation
Can detect presence based on ability to deplete
complement from serum (complement fixation)
Antigen added to serum with complement
If antibodies against antigen present, activates and
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depleted complement
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Steps of CFT
CFT::
1. Heat inactivate the test serum (to
detect presence or absence of Ab) to
get rid of the native complement
complement.. (560
C for 30 minutes)
2. Then add measured amounts of Ag
(known) and complement (known), to
the serum (unknown Ab)
Ab)..
3. If Ab specific for the known Ag is
present in the serum, Ag
Ag--Ab complexes
will form and bind all complement.
complement.
(reaction is invisible)
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Steps
Steps
Results::
Results
If all of the complement has been fixed, none
will be free to lyse the sheep rbcs
rbcs.. (No
hemolysis, indicates a positive complement
fixation test
test;; positive for the unknown Ab in
the serum)
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Interpretation of CFT
If no Ab is present in the patients serum, the
complement is not fixed and is free to interact in the
indicator system and lyse the rbcs.
rbcs. (Hemolysis
indicates a negative test;
test; negative for the unknown
Ab in the patients serum
serum.. The only things reacting
are the knowns
knowns..)
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Neg
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Complement Fixation
Methodology
Ag mixed with test serum to be assayed
for Ab
Erythrocytes coated with Abs is added
Amount of erythrocyte lysis is determined
No Ag
Ag
Ag
Patients
serum
Ag
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Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
EnzymeAssays (ELISA)
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Detection principles
Radiolabelled isotopes
125I,
Enzymes
Peroxydase
Chromophores
Fluorogenic probes, fluorescent proteins
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Rosalyn S. Yalow
American physicist who won
the
Nobel
prize
for
development
of
radioimmunoassays of peptide
hormones
The process made it possible
to detect and measure minute
amounts of hormones, drugs,
enzymes, and antibodies
The introduction of radio
radio-immunoassay is probably the
single
most
important
advance
in
biological
measurement of the past two
decades.. It has revolutionized
decades
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Prof. Md. Akram, MMC
one major discipline and
influenced several others
others..
47
Improved Diagnostics
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ELISA Formats
Direct sandwich ELISA antibodies (Ab) are
coated to micro wells.
wells. Antigen (Ag) is added and
binds with antibody.
antibody. Excess antigen is washed away
away..
Enzyme conjugate (Ab
(Ab--E) is added and binds with
antigen to form the double antibody sandwich
sandwich.. Wells
are washed to remove any excess (Ab
(Ab--E)
E).. Substrate is
added and color development is observed.
observed. The
enzyme conjugate binds directly to the antigen.
antigen.
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Ab
Ag
Ab--E
Ab
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Types of ELISA
Three different methods used to perform ELISAs
Direct method (different from book)
Indirect method
Capture method (called direct method
in book)
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Indirect ELISA
Antigen attached to well
Primary antibody added, unbound antibody removed
Enzyme conjugated secondary antibody added,
recognizes primary antibody
Unbound secondary antibody removed
Substrate for enzyme conjugated to secondary
antibody added
Color develops only if primary antibody bound
More sensitive than direct ELISA, does not require
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Prof. Md. Akram, MMC
production
of numerous conjugated
antibodies
53
Capture ELISA
Antibody attached to well
Sample added to well, antigen captured by antibody
Enzyme conjugated second antibody against antigen
added to well - may be against second epitope or same
epitope as antibody used to capture antigen
Unbound antibody removed
Substrate added, color develops if antigen present in
sample applied to well
Useful for detecting antigens present as very minor
species in sample
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Elisa: Enzyme
Enzyme--linked immunosorbent assay
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Sandwich Elisa
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Components
Enzyme::
Enzyme
Alkaline phosphatase
Horse radish peroxidase
Substrate :
Hydrogen peroxide
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Labeled
Anti-Ig
Ab in
Patients
sample
Immobilized
Ag
Solid
Phase
Quantitative
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Labeled
Ab
Ag in
Patients
sample
Ag
Immobilized
Solid
Phase
Quantitative
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Prior to Test
+
Labeled
Ag
Test
Use predetermined
amounts of labeled Ag
and Ab and add a sample
containing unlabeled Ag
as a competitor
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Labeled
Ag
+
Patients
sample
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Test
+
Solid Labeled
Ag
Phase
+
Patients
sample
Solid
Phase
Quantitative
Most sensitive test
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Immunofluorescence
Direct
Ab to tissue Ag is labeled with fluorochrome
Fluorescen isothiocyanate (FITC), Tetramethy
Rhodamine isothiocyanate (TRITC)
Fluorochrome
Labeled Ab
Ag
Tissue Section
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Immunofluorescence
Indirect
Ab to tissue Ag is
unlabeled
Fluorochrome
Fluorochrome--labeled
anti--Ig is used to
anti
detect binding of the
first Ab.
Unlabeled
Ab
Fluorochrome
Labeled Anti-Ig
Ag
Tissue Section
Qualitative to SemiQuantitative
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Immunofluorescence
Flow Cytometry
Cells in suspension are labeld with fluorescent tag
Direct or Indirect Fluorescence
Cells analyzed on a flow cytometer
Flow
Tip
FL
Detector
Light
Scatter
Detector
Laser
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Immunofluorescence
Flow Cytometry cont.
Data displayed
Two Parameter Histogram
Number of Cells
Unstained cells
FITC-labeled cells
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