Download Antigen/ Antibody reactions Diagnostic Immunology

Antigen/ Antibody reactions
Diagnostic Immunology
Professor Md. Akram Hossain
MMC
12/21/13
Prof. Md. Akram, MMC
1
Types of antigenantigen- Antibody reactions in
vivo
1.
2.
3.
4.
5.
6.
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Agglutination
Precipitation
Complement fixation
Neutralization
Antibody dependant cell mediated
cytotoxicity (ADCC)
Immobilization
Prof. Md. Akram, MMC
2
Types of antigen antibody
reactions used in vitro
1.
2.
3.
4.
5.
6.
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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Prof. Md. Akram, MMC
3
Applications / use in vitro
Diagnosis of many diseases
Severity or stage of diseases
Respond to treatment
Epidemiology
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Prof. Md. Akram, MMC
4
How antigen – antibody reactions in vitro
helps in Dx?
Infectious disease
• By determining whether an individual has developed
antibodies in response to infection
infection..
• By detecting antigen of a particular infectious agent
from blood or other body fluids
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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Prof. Md. Akram, MMC
5
Which diseases can be diagnosed by
antigen-- antibody reactions?
antigen
Infectious diseases
• Bacterial
• Viral
• Protozoa
• Fungal
• Parasitic
Autoimmune diseases
Tumors
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Prof. Md. Akram, MMC
6
Other examples of how immunology can be
used in the diagnostic laboratory
Occasionally, bacteriology and viruses need to be
identified from cultures
cultures..
Positive cultures applied to slides can be examined
by immunofluorescence
immunofluorescence..
This is how we identify herpes simplex virus in
tissue culture and how we recognize the presence
of respiratory viruses in tissue culture.
culture.
Gonorrhoea and Legionella can be identified from
isolated colonies by the same method
Sometimes, specific antibodies can help to
determine the exact species
species..
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Prof. Md. Akram, MMC
7
What is the basis of AgAg- Ab
reactions?
Specificity between antigen and
antibody is the basis of diagnosis.
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Prof. Md. Akram, MMC
8
What are the limitations?
Cross
reaction
between
antigens/ antibodies
similar
Time for development of antibodies
against any infectious agent
Presence of antibodies even after
cure of disease
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Prof. Md. Akram, MMC
9
How antigen – antibody reactions in vitro helps
in Dx of infectious disease?
By determining whether an individual has
developed antibodies in response to
infection
• IgM antibodies are usually a reflection
of a recent infection.
infection.
• Rising levels of IgG antibodies often
indicate recent infection
• Sometimes a very high titre of antibody
will signal recent infection
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Prof. Md. Akram, MMC
10
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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Prof. Md. Akram, MMC
11
Types of Agglutination
Direct agglutination
1. Slide – Blood grouping, Serotyping of bacteria
2. Tube –Widal test (Classical)
Indirect or Passive agglutination
1.
2.
3.
4.
Hemagglutination
Latex agglutination
Particle agglutination
Co--agglutination
Co
Flocculation tests
Coombs test
•
•
12/21/13
Direct – to detect antibody bound to fetal
RBC surface
Indirect – To detect
circulating
antibody in
Prof. Md. Akram,
MMC
serum in mother
12
Advantages and disadvantages of
agglutination
Advantages
•
•
•
•
•
Most widely used
Very simple
No instrument is required
Cheap
Fairly sensitive
Disadvantages
• Not highly specific
• Not highly sensitive
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Prof. Md. Akram, MMC
13
Direct agglutination
Occurs when the
antigenic
determinant
is
inherent
to
the
particle
itself..
itself
(naturally)
Example #1 – Using
group A rbc’s to
detect
antianti-A
in
serum..
serum
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Prof. Md. Akram, MMC
14
Direct agglutination..2
Example # 2 – Using
bacteria (Ag) looking
for Ab in serum.
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Indirect or Passive agglutination
Results
when
inert
particles are coated
with soluble Ags which
may react with Ab
Ab..
Particles include latex,
rbc’s, charcoal, etc
etc..
Example – Ag attached
to
latex
particle
(known)
+
serum
looking for (unknown)
Ab.. If Ab present, you
Ab
get
visible
agglutination..
agglutination
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Prof. Md. Akram, MMC
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Passive
Agglutination/Hemagglutination
Definition - agglutination test done
with a soluble antigen coated onto
a particle
+
• Applications
12/21/13
– Measurement of antibodies to soluble antigens
Prof. Md. Akram, MMC
17
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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Prof. Md. Akram, MMC
18
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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Prof. Md. Akram, MMC
19
Latex agglutination
Latex particles can be
coated with Ab, and in
the presence of Ag
can
form
visible
aggregates..
aggregates
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Prof. Md. Akram, MMC
20
Hemagglutination
Agglutination of rbc’s
as a result of Ab
interaction
with
antigenic determinants
on rbc’s surfaces
surfaces..
Example
–
using
group A rbc’s to detect
anti--A in serum.
anti
serum.
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Coombs (Antiglobulin)Tests
• Incomplete Ab
• Direct Coombs Test
– Detects antibodies on erythrocytes
+
Patient’s RBCs
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Coombs Reagent
(Antiglobulin)
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22
Coombs (Antiglobulin)Tests
Indirect Coombs Test
• Detects anti
anti--erythrocyte antibodies in
serum
Step 1
+
Patient’s
Serum
Target
RBCs
Step 2
+
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Coombs Reagent
Prof. Md. Akram, MMC
(Antiglobulin)
23
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 & RPR’s.
RPR’s.
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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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Advantages and disadvantages of
precipitation
Advantages
• Fairly sensitive
• High specificity
Disadvantages
• Time consuming
• Some costly instruments are required
• High technical skill required
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Prof. Md. Akram, MMC
29
Radial Immunodiffusion (Mancini)
• Method
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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Prof. Md. Akram, MMC
30
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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Prof. Md. Akram, MMC
– Precipitin arc represent
individual antigens
31
Immunoelectrophoresis
Method
Interpretation
Qualitative
• Relative concentration
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Prof. Md. Akram, MMC
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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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Prof. Md. Akram, MMC
33
Complement fixation test
(CFT)
Lattice formation not required
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34
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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Complement Fixation Reaction
• Antibody
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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Prof. Md. Akram, MMC
38
Steps…
Steps
…
• If Ab (unknown) specific for the known Ag is
not present in the serum, then the known Ag
and complement remain unbound
unbound..
• Indicator system
system:: add sheep rbc’s coated with
known Ab specific for known Ag
Ag..
Results::
Results
• If all of the complement has been fixed, none
will be free to lyse the sheep rbc’s
rbc’s.. (No
hemolysis, indicates a positive complement
fixation test
test;; positive for the unknown Ab in
the serum)
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Prof. Md. Akram, MMC
39
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 rbc’s.
rbc’s. (Hemolysis
indicates a negative test;
test; negative for the unknown
Ab in the patients serum
serum.. The only things reacting
are the knowns
knowns..)
• Ag/Ab/C + AbAb-coated rbc’s = no hemolysis
(positive)
• Ag/C + Ab
Ab--coated rbc’s = hemolysis
(negative)
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Prof. Md. Akram, MMC
40
Complement fixation test
Pos
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Neg
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Advantages and disadvantages of CFT
Uses
• CFT for kalazar, Filaria, Gonoccal CFT
• CFT for many viral infections
Advantages
• Fairly sensitive
• Wide application
application-- can be used for variety of
diseases
Disadvantages
• Time consuming
• Very difficult to standardize
• High technical skill required
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Prof. Md. Akram, MMC
42
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
Patient’s
serum
Ag
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43
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
EnzymeAssays (ELISA)
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44
Detection principles
Radiolabelled isotopes
• 125I,
14C, 32P, 35S
Enzymes
• Peroxydase
Chromophores
• Fluorogenic probes, fluorescent proteins
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45
Nobel Prize Winners
Rosalyn YalowYalowdiscovered radio –immunoimmunoassay (RAI) by studying the
reaction of insulin with
antibodies
• Presented to the world in
1959
(Dash 55)
• RIA used in endocinology,
virology (Dash 56)
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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
Radioimmunoassay: A very sensitive,
Radioimmunoassay:
specific laboratory test (assay) using
radiolabeled (and unlabeled) substances in
an
immunological
(antibody--antigen)
(antibody
reaction..
reaction
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48
RIA: radio immuno assay
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49
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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50
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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51
Direct ELISA (According to Dr. Nika)
• Antigen attached to well
• Unbound antigen removed by washing
• Enzyme conjugated antibody added to well
• Unbound antibody washed away
• Substrate to enzyme conjugated to antibody added
• If antibody bound, substrate is cleaved
• Color develops, allows identification of organism
• Requires production of conjugated antibody for each
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Prof. Md. Akram, MMC
bacterial
species
52
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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Prof. Md. Akram, MMC
54
Elisa: Enzyme
Enzyme--linked immunosorbent assay
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55
Sandwich Elisa
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Components
Enzyme::
Enzyme
•Alkaline phosphatase
•Horse radish peroxidase
•Substrate :
•Hydrogen peroxide
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57
Solid Phase NonNon-Competitive
RIA/ELISA
Ab detection
• Immobilize Ag
• Incubate
with
sample
• Add labeled anti
anti-Ig
• Amount of labeled
Ab
bound
is
proportional
to
amount of Ab in
the sample
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Labeled
Anti-Ig
Ab in
Patient’s
sample
Immobilized
Prof. Md. Akram, MMC
Ag
Solid
Phase
• Quantitative
58
Solid Phase NonNon-Competitive
RIA/ELISA
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
Labeled
Ab
Ag in
Patient’s
sample
Ag
Immobilized
Solid
Phase
• Quantitative
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Prof. Md. Akram, MMC
59
Competitive RIA/ELISA for Ag
Method
• Determine amount
of Ab needed to
bind to a known
amount of labeled
Ag
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
Prof. Md. Akram, MMC
+
+
Patient’s
sample
60
Competitive RIA/ELISA for Ag
Method cont.
• Determine
amount of
labeled Ag
bound to Ab
Test
+
Solid Labeled
Ag
Phase
+
+
Patient’s
sample
Solid
Phase
– Concentration determined from a standard curve
using known amounts of unlabeled Ag
• Quantitative
– Most sensitive test
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Prof. Md. Akram, MMC
61
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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62
Direct Immunofluorescent Test
• Requires production of species specific antibody
• Fluorescent group (FITC) directly conjugated to species specific
antibody
• Bacteria attached to slide, antibody added to bacteria, unbound
antibody removed
• Bacteria observed at wavelength of light that causes conjugate to
fluoresce
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63
Indirect Immunofluorescent Test
• Primary antibody added to specimen, unbound washed away
• Secondary conjugated antibody added, recognizes primary
antibody
• Unbound secondary antibody removed, specimen observed at
wavelength of light that produces fluorescence
• More sensitive, secondary antibody amplifies signal
• Also more time consuming
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Prof. Md. Akram, MMC
64
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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65
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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Prof. Md. Akram, MMC
66
Immunofluorescence
• Flow Cytometry cont.
– Data displayed
Two Parameter Histogram
Number of Cells
Unstained cells
FITC-labeled cells
Green Fluorescence Intensity
One Parameter Histogram
Red Fluorescence Intensity
Green Fluorescence Intensity
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67
use the cellular immune response to
diagnose infections?
Skin testing is used most often (the TB
skin test is the most common) (Mantoux
test)
• TB antigens are injected under the skin (5
TU)
• Over 48 hours, cells migrate towards the
injected antigen
• This produces local swelling (induration).
(induration). The
diameter of the induration is measured
measured..
• Individuals without past TB have no
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Prof. Md. Akram, MMC
induration
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