Ag-Ab Reactions in Diagnosis

Antigen/ Antibody reactions
Diagnostic Immunology
Professor Md. Akram Hossain
MMC
12/21/13
Prof. Md. Akram, MMC
Types of antigenantigen- Antibody reactions in

vivo
1.
2.
3.
4.
5.
6.
12/21/13
Agglutination
Precipitation
Complement fixation
Neutralization
Antibody dependant cell mediated
cytotoxicity (ADCC)
Immobilization
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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Applications / use in vitro

Diagnosis of many diseases
Severity or stage of diseases
Respond to treatment
Epidemiology
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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
12/21/13
Which diseases can be diagnosed by

antigen-- antibody reactions?
antigen
Infectious diseases
Bacterial
Viral
Protozoa
Fungal
Parasitic
Autoimmune diseases
Tumors
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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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What is the basis of AgAg- Ab

reactions?
Specificity between antigen and
antibody is the basis of diagnosis.
12/21/13
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
12/21/13
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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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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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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13
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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14
Direct agglutination..2
Example # 2 Using
bacteria (Ag) looking
for Ab in serum.
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15
Indirect or Passive agglutination

Results
when
inert
particles are coated
with soluble Ags which
may react with Ab
Ab..
Particles include latex,
rbcs, 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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16
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

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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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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19
Latex agglutination
Latex particles can be
coated with Ab, and in
the presence of Ag
can
form
visible
aggregates..
aggregates
12/21/13
20
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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21
Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes
+
Patients RBCs
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Coombs Reagent
(Antiglobulin)
22
Indirect Coombs Test
Detects anti
anti--erythrocyte antibodies in
serum
Step 1
+
Patients
Serum
Target
RBCs
Step 2
+
12/21/13
Coombs Reagent
(Antiglobulin)
23
Applications
Detection of anti
anti--Rh Ab
Autoimmune hemolytic anemia
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24
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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25
12/21/13
26
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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27
Types of precipitation
1.
Precipitation in gel
Single radial immunodiffusion
Double diffusion
2.
Precipitation in Electrophoresis
Immune electrophoresis
Counter current Immune
electrophoresis (CIE)
12/21/13
28
Advantages and disadvantages of

precipitation
Advantages
Fairly sensitive
High specificity
Disadvantages
Time consuming
Some costly instruments are required
High technical skill required
12/21/13
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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30
Immunoelectrophoresis
Method
Ags are separated by electrophoresis
Ab is placed in trough cut in the agar
Ag
Ag
Ab
Ag
Ab
Interpretation
12/21/13

Precipitin arc represent
individual antigens
31
Immunoelectrophoresis
Method
Interpretation
Qualitative
Relative concentration
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32
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
12/21/13
33
Complement fixation test

(CFT)
Lattice formation not required
12/21/13
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
12/21/13
35
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
12/21/13
depleted complement
36
12/21/13
37
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)
12/21/13
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 rbcs 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 rbcs
rbcs.. (No
hemolysis, indicates a positive complement
fixation test
test;; positive for the unknown Ab in
the serum)
12/21/13
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 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..)
Ag/Ab/C + AbAb-coated rbcs = no hemolysis

(positive)
Ag/C + Ab
Ab--coated rbcs = hemolysis
(negative)
12/21/13
40
Complement fixation test

Pos
12/21/13
Neg
41
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
12/21/13
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
Patients
serum
Ag
12/21/13
43
Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
EnzymeAssays (ELISA)
12/21/13
44
Detection principles
Radiolabelled isotopes
125I,
14C, 32P, 35S
Enzymes
Peroxydase
Chromophores
Fluorogenic probes, fluorescent proteins
12/21/13
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)
12/21/13
46
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
12/21/13
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
12/21/13
48
RIA: radio immuno assay
12/21/13
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.
12/21/13
Ab
Ag
Ab--E
Ab
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)
12/21/13
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
12/21/13
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
12/21/13
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
12/21/13
54
Elisa: Enzyme
Enzyme--linked immunosorbent assay
12/21/13
55
Sandwich Elisa
12/21/13
56
Components
Enzyme::
Enzyme
Alkaline phosphatase
Horse radish peroxidase
Substrate :
Hydrogen peroxide
12/21/13
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
12/21/13
Labeled
Anti-Ig
Ab in
Patients
sample
Immobilized
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
Patients
sample
Ag
Immobilized
Solid
Phase
Quantitative
12/21/13
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
12/21/13
+
Labeled
Ag
+
Patients
sample
60
Competitive RIA/ELISA for Ag

Method cont.
Determine
amount of
labeled Ag
bound to Ab
Test
+
Solid Labeled
Ag
Phase
+
Patients
sample
Solid
Phase
Concentration determined from a standard curve

using known amounts of unlabeled Ag
Quantitative
Most sensitive test
12/21/13
61
Immunofluorescence
Direct
Ab to tissue Ag is labeled with fluorochrome
Fluorescen isothiocyanate (FITC), Tetramethy
Rhodamine isothiocyanate (TRITC)
Fluorochrome
Labeled Ab
Ag
Tissue Section
12/21/13
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
12/21/13
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
12/21/13
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
12/21/13
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
12/21/13
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

12/21/13
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
12/21/13
68
induration
12/21/13
69

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Antigen/ Antibody reactions

Prof. Md. Akram, MMC

Types of antigenantigen- Antibody reactions in

Types of antigen antibody

Prof. Md. Akram, MMC

Applications / use in vitro

Prof. Md. Akram, MMC

How antigen antibody reactions in vitro

Prof. Md. Akram, MMC

Which diseases can be diagnosed by

Prof. Md. Akram, MMC

Other examples of how immunology can be

Prof. Md. Akram, MMC

What is the basis of AgAg- Ab

Prof. Md. Akram, MMC

What are the limitations?

Time for development of antibodies

Prof. Md. Akram, MMC

How antigen antibody reactions in vitro helps

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Indirect or Passive agglutination

Direct to detect antibody bound to fetal

Advantages and disadvantages of

Most widely used

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Indirect or Passive agglutination

Prof. Md. Akram, MMC

Measurement of antibodies to soluble antigens

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Advantages and disadvantages of

Prof. Md. Akram, MMC

Radial Immunodiffusion (Mancini)

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Complement fixation test

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Complement Fixation Reaction

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

If Ab (unknown) specific for the known Ag is

Prof. Md. Akram, MMC

Ag/Ab/C + AbAb-coated rbcs = no hemolysis

Prof. Md. Akram, MMC

Complement fixation test

Prof. Md. Akram, MMC

Advantages and disadvantages of CFT

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC

Prof. Md. Akram, MMC