Antigen/ Antibody reactions

Diagnostic Immunology

Professor Md. Akram Hossain

MMC

12/21/13 Prof. Md. Akram, MMC 1

Types of antigen-
antigen- Antibody reactions in
vivo
1. Agglutination
2. Precipitation
3. Complement fixation
4. Neutralization
5. Antibody dependant cell mediated
cytotoxicity (ADCC)
6. Immobilization

12/21/13 Prof. Md. Akram, MMC 2

 Types of antigen antibody
reactions used in vitro
1. Agglutination
2. Precipitation
3. Neutralization
4. Complement fixation
5. Fluorescent--antibody technique
Fluorescent
6. ELISA-- Enzyme linked immunosorbent
ELISA
assay
7. Radio immunoassay
8. ImmunochromatographY (ICT)
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 Applications / use in vitro
Diagnosis of many diseases
Severity or stage of diseases
Respond to treatment
Epidemiology

12/21/13 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
12/21/13 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

12/21/13 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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 What is the basis of Ag-
Ag- Ab
reactions?

Specificity between antigen and

antibody is the basis of diagnosis.

12/21/13 Prof. Md. Akram, MMC 8

 What are the limitations?

Cross reaction between similar

antigens/ antibodies

Time for development of antibodies

against any infectious agent

Presence of antibodies even after

cure of disease
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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

12/21/13 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..

12/21/13 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. Hemagglutination
2. Latex agglutination
3. Particle agglutination
4. Co--agglutination
Co
Flocculation tests
Coombs test
• Direct – to detect antibody bound to fetal
RBC surface
•
12/21/13 Indirect – To detect circulating
Prof. Md. Akram, MMC antibody in 12

serum in mother
 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

12/21/13 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 anti-
anti-A in
serum..
serum
12/21/13 Prof. Md. Akram, MMC 14
 Direct agglutination..2

Example # 2 – Using
bacteria (Ag) looking
for Ab in serum.

12/21/13 Prof. Md. Akram, MMC 15

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

12/21/13 Prof. Md. Akram, MMC 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
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

12/21/13 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

12/21/13 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

12/21/13 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.

12/21/13 Prof. Md. Akram, MMC 21

 Coombs (Antiglobulin)Tests

• Incomplete Ab
• Direct Coombs Test
– Detects antibodies on erythrocytes

Patient’s RBCs Coombs Reagent

(Antiglobulin)

12/21/13 Prof. Md. Akram, MMC 22

 Coombs (Antiglobulin)Tests
Indirect Coombs Test
• Detects anti
anti--erythrocyte antibodies in
serum
Step 1
+
Patient’s Target
Serum RBCs

Step 2

+
Coombs Reagent
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(Antiglobulin)
Prof. Md. Akram, MMC
 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.

12/21/13 Prof. Md. Akram, MMC 25

12/21/13 Prof. Md. Akram, MMC 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
optimum concentration, lattice
formation occurs
occurs..

12/21/13 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 29

 Radial Immunodiffusion (Mancini)

Ab in gel
• Method
– Ab in gel Ag Ag Ag Ag

– Ag in a well
Interpretation
• Diameter of ring
is proportional
to the Diameter2
concentration
Quantitative
• Ig levels
Ag Concentration
12/21/13 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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– Precipitin arc represent individual antigens
 Immunoelectrophoresis
Method
Interpretation
Qualitative
• Relative concentration

12/21/13 Prof. Md. Akram, MMC 32

 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
12/21/13 Prof. Md. Akram, MMC 33
 Complement fixation test
(CFT)
Lattice formation not required

12/21/13 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 36

depleted complement
12/21/13 Prof. Md. Akram, MMC 37
Steps of CFT
CFT::

1. Heat inactivate the test serum (to

detect presence or absence of Ab) to
complement.. (560
get rid of the native complement
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 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)

12/21/13 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 + Ab-
Ab-coated rbc’s = no hemolysis
(positive)
• Ag/C + Ab Ab--coated rbc’s = hemolysis
(negative)
12/21/13 Prof. Md. Akram, MMC 40
 Complement fixation test
Pos Neg

12/21/13 Prof. Md. Akram, MMC 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 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

Ag No Ag
Ag
Patient’s
serum
Ag

12/21/13 Prof. Md. Akram, MMC 43

 Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
Enzyme-
Assays (ELISA)

12/21/13 Prof. Md. Akram, MMC 44

 Detection principles

Radiolabelled isotopes
• 125I, 14C, 32P, 35S

Enzymes
• Peroxydase
Chromophores
• Fluorogenic probes, fluorescent proteins

12/21/13 Prof. Md. Akram, MMC 45

 Nobel Prize Winners
Rosalyn Yalow-
Yalow-
discovered radio –immuno-
immuno-
assay (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 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 47
one major discipline and
influenced several others
others..”
 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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 RIA: radio immuno assay

12/21/13 Prof. Md. Akram, MMC 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
Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 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

bacterial
12/21/13
species Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 53
production of numerous conjugated antibodies
 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

12/21/13 Prof. Md. Akram, MMC 55

 Sandwich Elisa

12/21/13 Prof. Md. Akram, MMC 56

 Components
Enzyme::
Enzyme
•Alkaline phosphatase
•Horse radish peroxidase

•Substrate :
•Hydrogen peroxide

12/21/13 Prof. Md. Akram, MMC 57

 Solid Phase Non-
Non-Competitive
RIA/ELISA
Ab detection
Labeled
• Immobilize Ag Anti-Ig
Ab in
• Incubate with Patient’s
sample sample
• Add labeled anti
anti-- Immobilized Ag
Ig
• Amount of labeled Solid
Phase
Ab bound is
proportional to
amount of Ab in
the sample • Quantitative
12/21/13 Prof. Md. Akram, MMC 58
 Solid Phase Non-
Non-Competitive
RIA/ELISA
Ag detection
Labeled
• Immobilize Ab Ab
• Incubate with sample Ag in
Patient’s
• Add labeled antibody sample Ag
• Amount of labeled Ab Immobilized
bound is proportional
to the amount of Ag Solid
Phase
in the sample
• Quantitative
12/21/13 Prof. Md. Akram, MMC 59
 Competitive RIA/ELISA for Ag
Method
• Determine amount Prior to Test
of Ab needed to
bind to a known
amount of labeled
+
Ag Labeled
Ag
Test

• Use predetermined
+ + +
amounts of labeled Ag
Labeled Patient’s
and Ab and add a sample Ag sample
containing unlabeled Ag
as a competitor
12/21/13 Prof. Md. Akram, MMC 60
 Competitive RIA/ELISA for Ag
Method cont.
• Determine
amount of Test
labeled Ag +
bound to Ab
+ +
Solid Labeled Patient’s Solid
Phase Ag sample Phase

– Concentration determined from a standard curve

using known amounts of unlabeled Ag
• Quantitative
– Most sensitive test
12/21/13 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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 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 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 64

 Immunofluorescence

Indirect
• Ab to tissue Ag is
Fluorochrome
unlabeled Labeled Anti-Ig
• Fluorochrome
Fluorochrome--labeled Unlabeled
Ab
anti--Ig is used to
anti
detect binding of the
first Ab. Ag
Tissue Section
• Qualitative to Semi-
Quantitative
12/21/13 Prof. Md. Akram, MMC 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 Prof. Md. Akram, MMC 66
 Immunofluorescence
• Flow Cytometry cont.
– Data displayed

One Parameter Histogram Two Parameter Histogram

Green Fluorescence Intensity

Unstained cells
Number of Cells

FITC-labeled cells

Green Fluorescence Intensity Red Fluorescence Intensity

12/21/13 Prof. Md. Akram, MMC 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 induration Prof. Md. Akram, MMC 68
12/21/13 Prof. Md. Akram, MMC 69