GOOD

MORNING

Dr. SWETHA
RAGHOJI
DEPT OF ORAL & MAXILLO
FACIAL PATHOLOGY
2

INTRODUCTION.
APPLICATIONS .
FEATURES OF ANTIGEN ANTIBODY
REACTION.
ANTIGEN ANTIBODY REACTION
a) PRECIPITATION REACTIONS.
b) IMMUNODIFFUSION.
c) AGGLUTINATION REACTIONS.
d) IMMUNOFLUORESCENCE.

e)COMPLEMENT FIXATION TEST

f)RADIOIMMUNOASSAY
g)ENZYME IMMUNOASSAY
h)IMMUNOELECTROBOLT TECHNIQUE

A reaction that occurs when an antigen

combines with a corresponding antibody to
produce an immune complex.

The interaction of an antibody with an

antigen has been described as similar to a
lock and key concept.

Antibody combining site and the part of the

antigen to which it binds must be
complementary, so that van der Waal's and
hydrophobic forces can stabilize the
interaction between the antigen and
antibody.
5

Applications

In the body or in vivo

It forms the basis of immunity against
infectious diseases.

In the laboratory or in vitro

For diagnosis of infections.
Detection & quntitation of either
antigens or antibodies.
6

1.

The reaction is specific, an antigen combining

only with its homologous antibody & vice versa.

2.

Entire molecules of Ag & Ab react and not the

fragments.

3.

There is no denaturation of the antigen or the

antibody during the reaction.

4.

The combination is firm but reversible. The

firmness of the union is influenced by the
affinity & avidity of the reaction.
7

5.Both antigens & antibody participate in

the formation of agglutinates.
6.Antigens & antibodies can combine in
varying proportions.

Ag Ab reaction occur in three stages.

Primary stage:
It is the initial reaction between the antigen &
antibody.
The reaction is rapid ,occurs even at low temp.
The reaction is reversible.
It can be detected by estimating free & bound
antigen or antibody separately in the reaction
mixture.

Secondary stage:

Demonstrable events such as

precipitation, agglutination, lysis of cells,
fixation of complement takes place.

Tertiary stage:

Some antigen antibody reactions

occurring in vivo initiate chain reactions
that lead to neutralization or destruction
of injurious agents or tissue damage.
10

Specificity: The ability of the test to

detect reactions between homologous
antigens and antibodies only & with no
other.

Sensitivity: The ability of the test to

detect even very minute quantities of
antigen or antibody.

11

When a soluble antigen

combines with its antibody in the
presence of electrolyte the Ag-Ab
complex
forms an insoluble precipitate.

Precipitate remains suspended

as floccules , the reaction is
known as flocculation.

precipitation can take place in

liquid media or in gel.
12

MARRACK (1934) proposed the

lattice hypothesis to explain the
mechanism of precipitation.

Multivalent antigens combine with

bivalent antibodies in varying
proportions ,depending on the AgAb in the reacting mixture.

Precipitation results when a large

lattice is formed consisting of
alternating Ag & Ab molecules.
13

Identification of bacteria.
Detection of antibody for diagnostic
purposes.
Forensic application in identification of
human blood.
To standardise toxins & antitoxins.

14

Ring test:

It was first introduced by Ascoli

in 1902.
Simplest type precipitation test.
Layering the antigen solution
over a column of antiserum in a
narrow tube.
Precipitate forms at the junction
of two liquids, which denotes a
positive result.
Ex- Ascolis test

15

Slide test:-

Drop of antigen
solution is added
to a drop of
inactivated
patients serum on
a slide & mixed by
shaking, floccules
appear.
Ex:-VDRL Test
16

Tube test:
Tube flocculation test is employed for
the standardisation of toxins & toxoids.
Serial dilutions of the toxin or toxiod
are added to the tubes containing a
fixed quantity of the antitoxin.
The amount of toxin or toxiod that
flocculates optimally with one unit of
the antitoxin is defined as an Lf dose.
17

Immunodiffusion tests are precipitation

tests done in gel media.

Advantages in allowing precipitation in a

gel rather than liquid are:-

Reaction is visible as a distinct band of

precipitation.
Reaction is stable.
The number of different antigens in the
reacting mixture can be readily observed.

18

Single diffusion in one dimension:-

Antibody is incorporated in a agar gel & the

antigen solution is layered over it in a test
tube.

The antigen diffuses downward through the

agar gel, forming a line of precipitation .

The number of bands indicates the number

of different antigens present.

It is single diffusion of antigen only & in one

dimension i.e towards antibody in agar gel
19

The antibody is incorporated

in gel, above which is placed a
column of plain agar.

Antigen is layered on top of

plain agar.

Antigen antibody move

towards column of plain agar
& forms a band of precipitate.
20

The antiserum is
incorporated in agar gel on
a petridish.
The wells are cut on the
surface of gel.
The antigen is placed in a
well cut in an agar gel
containing suitable diluted
antibody.
A ring of precipitate forms
where the reactants meet in
optimal proportions.

Ab in gel

Ag

Ag

Ag

Ag

21

The higher is the

concentration of the
examined antigen, the
greater is the diameter of
the ring.
According to the diameter
of the ring it is possible to
count the concentration of
the examined antigen.
This type of
immunodiffusion is used
for quantitative
determination of
immunoglobulins.
22

Double immunodiffusion

Most widely used method .

Principle:- In this technique Ag & Ab

are allowed to diffuse in the agar
medium , when these two meet in
optimal proportions a precipitation
line is formed.
Method:- Agar gel is poured on a slide
& wells are cut using a template.

Antiserum is placed in the central

well & different antigens in the
surrounding wells.
23

Two types of reactions are noticed if

Ag are placed in adjacent wells.

Type 1 The reaction of identity

If two adjacent antigens are identical
the lines of precipitate will fuse.

Type 2-The reaction of non identity

If they are unrelated they will cross
each other.
This reaction is used for diagnosing
various bacterial, viral, fungal and
autoimmune diseases.
24

Grabar and williams devised the technique of

immunoelectrophoresis.
Is a combination of electrophoresis and
gel-diffusion precipitation.
Antigens (most usually serum proteins) are first
divided by electrophoresis according to their electric
charge (albumins are directed towards the anode
and globulins towards the cathode) on an agar
coated slide.
After electrophoresis is finished the longitudinal
troughs are cut in the agar parallel to the axis of
electrophoresis and filled with antibody.
-

+
Ag

Ag
Ab

25

Diffusion then takes place.

When antigen and antibody meet
precipitate lines of single immunoglobulin
classes occur.
The resulting lines are photographed &
the slides are dried and read after staining
This is useful for testing for normal &
abnormal proteins in serum & blood.
Ag

Ab
26

Is a rapid and more sensitive variant of double

diffusion method.

The principle of this test procedure is that Ag and

Ab migrate toward each other by electrophoresis
in opposite direction resulting in precipitation at a
point between them.

The clinical application are for detecting various

antigens such as alphafetoprotein in serum &
specific antigens of cryptococcus &
+
meningococcus- in the CSF.
Ag

Ab

27

When a particulate
antigen is mixed with its
antibody in the presence
of electrolytes at a
suitable temperature & pH
the particles are clumped
or agglutinated.

Lattice hypothesis holds

good for agglutination
also.
28

A drop of antiserum is
added to smooth, uniform
suspension of antigen in a
saline on a slide,
agglutination takes place.

A positive result is
indicated by the clumping
of the particles.
29

It is neccesary to have a control on the

same slide consisting of antigen
suspension in saline, to ensure that
antigen is not autoagglutinable.

Slide agglutination is a routine procedure

for the identification of many bacterial
isolates from clinical specimens.

It is also the method used for blood

grouping & cross-matching.
30

Standard quantitative method for the

measurement of antibodies.
Fixed volume of antigen suspension is
added to an equal volume of serial
dilutions of an antiserum in test tubes,
the agglutination titre of the serum is
estimated.
Tube agglutination is routinely
employed for serological diagnosis of
typhoid, typhus fever.
31

The antiglobulin test was devised by

Coombs, Mourant,& race for detection
of anti-Rh antibodies that do not
agglutinate Rh +ve erythrocytes.

32

When sera containing incomplete anti

Rh Ab are mixed with coressponding Rh
positive erythrocytes the incomplete
Ab globulin coats the erythrocytes but
no agglutination occurs.
when such coated erythrocytes are
treated with antiglobulin or coombs
serum (rabbit antiserum against
human gamma globulin) the cells are
agglutinated
33

When antibodies bind to erythrocytes, they do not

always result in agglutination.
This can result from the antigen antibody ratio
being in antigen excess or antibody excess
preventing the effective cross linking of the cells.
A second antibody directed against the antibody
coating the red cells. This anti-immunoglobulin can
now cross link the red blood cells and result in
agglutination.

Patients RBCs

Coombs Reagent

For detection of anti Rh antibodies.

For demonstration of any type of
incomplete antibody .

35

Passive agglutination test:

Soluble antigens attaching to the surface

of the carrier particles, it is possible to
convert precipitation tests in to
agglutination tests which are more
convenient & more sensitive for the
detection of antibodies.
+

36

It was first described by Coons & his

colleagues.

Immunofluorescence is a technique whereby an

antibody labeled with a fluorescent molecule is
used to detect the presence of an antigen by
the fluorescence emitted by the bound
antibody.

Direct
Indirect

a. Direct Immunofluorescence

In direct immunofluorescence,
the antigen is fixed to the
slide.
Fluroescein labeled antibodies
are added to the slide &
incubated .
Slide is washed to remove
any unbound Ab & examined
fluoresence microscope.
Iff test is positive fluorescence
occurs at the combinig site.

Fluorochrome
Labeled Ab

Ag

38

Routinely used as a method of

diagnosing rabies.

Disadv :-separate fluorescent

conjugates have to be prepared
against each antigen to be tested.

39

b. Indirect Immuno
fluorescence
A known antigen is fixed on a
slide.
The unknown Ab is applied to
the slide.
If antibody is present in the
serum it attaches to known
antigen on the slide.
For detection of this Ag Ab
reaction, fluorescein tagged Ab
to human globulin is added.
If test is positive fluorescence
occurs at the combinig site.

Fluorochrome
Labeled AntiIg
Unlabeled
Ab

Ag

40

Applications

Direct immunofluorescence has more

applications in dentistry than indirect
immunofluorescence .
Direct technique has applications in
diagnosis of various lesions such as
lupus erythematous, pemphigus
vulgaris, lichen planus.
41

It is the process in which measurements

are made while cells in a liquid
suspension are forced to flow one at a
time through a measuring device.

Counting & examining cells suspended in

stream of fluid.

Allows simultaneous analysis of the

physical or chemical characteristics of
single cells flowing through an optical or
electronic detection apparatus.
42

A beam of laser light is

directed onto a stream of
fluid containing suspension
of cells.
A number of detectors are
aimed at the point where
the stream passes through
the light beam.
Each cell passing through
the beam scatters the
light.
Scattered light is picked up
the detectors & analysed
by computers

43

By detecting variations in the

brightness at each detectors it is then
possible to extrapolate various types of
information about the physical &
chemical structure of individual cell.

44

1.

2.
3.
4.
5.

Used to support diagnosis of

malignancy when the morphological
changes are equivocal.
Used to classify tumors of borderline
malignancy.
Provides prognostic information
independent of stage & grade of tumor.
Helps to identify tumor relapse.
Helps to detect tissue of region of a
tumor.
45

COMPLEMENT FIXATION TEST

CFT is based on the principle of fixation

of complement factors to antigen
antibody complexes which is detected
by an indicator system consisting of
sheep RBC & antibodies to sheep RBC.

46

Method:-

Antigen is mixed with the test

serum to be assayed for
antibody and antigen antibody
complexes are allowed to form.
If no antigen antibody
complexes are present in the
tube, none of the complement
will be fixed.
However, if antigen antibody
complexes are present, they
will fix complement and
thereby reduce the amount of
complement in the tube.

After allowing
complement fixation by
any antigen antibody
complexes, a standard
amount of red blood
cells, which have been
pre-coated with antierythrocyte antibodies
is added.

If all the complement is

present , all the red
cells will be lysed.
48

If antigen antibody complexes are formed

some of the complement will be
consumed and thus when the antibodycoated red cells are added not all of them
will lyse.

49

By simply measuring the amount of red

cell lysis, by measuring the release of
hemoglobin into the medium, one can
indirectly quantitate antigen antibody
complexes in the tube.

Complement fixation tests are most

commonly used to assay for antibody in a
test sample.

50

Berson & Yallow in 1959.

RIA is a competitive
binding assay in which
fixed amounts of
antibody & radiolabelled
antigen react in the
presence of unlabelled
antigen.
The labelled &
unlabelled antigens
compete for the limited
binding sites on the
antibody.
51

This competition is determined by

the level of the unlabelled antigen
present in the reacting system.

After the reaction, the antigen is

seperated into free & bound
fractions and their radioactive
counts measured.

The concentration of the test

antigen can be calculated from
the ratio of the bound & total
antigen labels using a standard
dose response curve.
52

Enzyme labelled conjugates were first

introduced in 1966 .

In 1971 enzyme labelled antigens &

antibodies were developed as serological
reagents for the assay of antibodies &
antigens.

Versatility, sensititvity, simplicity,

economy & absence of radiation hazard
have made EIAs most widely used
procedure.
53

Enzyme immunoassay are based on

use of enzyme labelled immuno
reactants for detection of unlabelled
antibody or antigen.

EIAs are of two basic types:Homogeneous

Heterogeneous

54

In Homogeneous EIA, there is no need

to separate the bound & free fractions
so that the test can be completed in
one step.

55

Heterogeneous EIA requires the

separation of the free & bound
fractions.
It is multi step procedure, with
reagents added sequentially.
The major type of heterogeneous EIA is
Enzyme Linked Immunosorbent Assay.

56

Purified inactivated antigen is adsorbed onto

microassay plate wells.
Test serum is added to the well & incubated at
30 c for 30 min .
Well is then thoroughly washed.
If serum contains appropriate antibody, it will
form a stable complex with the antigen on the
57
plate.

secondary antibodies conjugated with substrate

specific enzyme is added. It conjugate with the
antibody if the test is positive.

Wash the plate, so that excess unbound enzymeantibody conjugates are removed.

substrate is added & after 30 min the colour that

develops is read by ELISA reader or
spectrophotometer.
58

"sandwich" ELISA, is used to detect antigen.

The steps are as follows:
A known quantity of antibody is bound to the
microassay plate.
Add the antigen-containing sample to the
plate.
Wash the plate, so that unbound antigen is
59
removed.

Add enzyme-linked secondary antibodies

which are specific to the primary antibodies.
Wash the plate, so that the unbound antibodyenzyme conjugates are removed.
substrate is added & after 30 min the colour
that develops is read using a microassay plate
reader
60

It has been used for detection of

antibodies.
The microtitre plates wells are coated
with antigen.
Sera to be tested is added to these wells
If Ab are present Ag Ab complex is
formed.
To detect this reaction enzyme labelled
specific Ab are added.
61

There is no Ag left for these Ab to act.

These Ab remain free & washed off.
Substrate is added but there is no
enzyme to act on it.
Therefore positive results show no
colour.

62

Detection of HIV antibodies in serum.

Detection of mycobacterial antibodies
in tuberculosis.
Detecion of roatvirus in faeces.

63

When antigens are first separated by poly

acrylamide gel electrophoresis &
transferred on to nitro cellulose paper
strips which are then used for detection of
antibodies in unknown sample, the
technique is known as immunoblotting

64

Method :- Western blotting is the

separation of differently charged
proteins via poly acrylamide gel
electrophoresis.

The separated proteins are

transferred to a sheet nitrocellulose
by placing the nitrocellulose directly
on the poly acrylamide gel, and
running a current perpendicularly
through the combination.
The nitrocellulose sheet is cut in
strips.

65

One strip is developed with a protein

stain to identify the position of all
the components.

Other strips are reacted with serum

suspected to have antibodies to the
protein of interest.

Unreacted antibodies are removed

by washing following which the strip
is incubated with fluorescent or
radio labeled antihuman antisera.

Visible colour bands indicate that

the patient serum contains
antibodies to the particular antigen.
66

This test is best known as the definitive

screen for anti-HIV antibodies.

67

Immunoelectronmicroscopy

When viral particles mixed with specific

anti sera are observed under the
electron microscope.
They are seen to be clumped .
Finds application in study of some
viruses such as hepatitis A virus.

68

Enzymes such as peroxidase can be

conjugated with anti bodies.
Tissue sections carrying the
corresponding antigens are treated
with peroxidase labled anti sera.
The peroxidase bound to the antigen
can be visualized under the electron
microscope.
69

THANK YOU

70

REFERENCES.

1.

Text book of Microbiology. R.

Ananthanarayana. 6th edition.
Microbiology For Dental Students.Rajesh
Bhatia 2nd edition.
Medical Microbiology &
Immunology.Warren Levinson 7th edition.
Text book of immunology. kuby .
Text of microbiology.prescott 6th edition.

2.
3.
4.
5.

71

Structure of antibody

72

Serial dilutions are done to determine

the antibody titre.

Interpretation of widal reaction

Titres of 1/100 or more for O
agglutinins.
Titres of 1/200 or more for H
agglutinins.

73

Amyloidosis
Leukemia
Plasma cell dyscrasias
Polycythemia

74

The lack of agglutinating activity of an

incomplete antibody may be due to to
restricted flexibility in the hinge region,
making it difficult for the antibody to
assume the required angle for optimal
cross linking of epitopes on two or
more particulate antigens.

75

Test controls are used to check whether

the faults are in the procedure which is
carried for the test or with the reagents
used.

76

A false positive is when there is no

disease but the results are positive.

A false negative is when there is

actually a disease but the results are
negative.

77

Radioactive iodine.
Tritium.

78

Flourescence in situ hybridization.

Probes can be hybridised to DNA contained

within chromosomes & immobilized on a
microscopic slide. This process is called in situ
hybridization.

The DNA is denatured & fixed on the microscopic

slide.

A fluorescent dye is applied & the slide is

exposed to a wavelength of light that excites the
fluorescence colour. This process is called FISH
79

The Ag particles are seen as small

fusiform needles which remain more or
less evenly dispersed in a non reactive
serum & aggregate into clumps in
reactive sera.

80

To detect whether any primary Ab are

bound in the wells, a second Ab that
recognizes human Ab is added.
If primary Ab are present the secondry
Ab will bind to them.

81

used to detect antibodies to specific

epitopes of electrophoretically
separated antigens.

82

Electrolytes reduces negative charges

on particles which help in aggregation.

83

Immuno suppression : involves an act

that reduces the activation or efficacy
of the immune system.
Immuno modulation: change in the
bodys immune system, caused by
agents that activate or supress its
function.

84

Gelatin ,pectin starch.

85

Single diffusion:
Determination of number of antigenic
substances such as blood plasma.
Double diffusion:
Used for diagnosis of certain fungal
infections such as histoplasmosis,
aspergillosis

86

A toxin of a pathogenic organism

treated so as to destroy its toxicity but
leave it capable of inducing the
formation of antibodies on injection.

87

It may lead to tissue injury in some

hypersensitivity reactions.

88

PCR is a revolutionary technique for

molecular genetic purpose with widespread
applications in diagnostics & research.
The technique is based on the principle that
a single strand of DNA has limitless capacity
to duplicate itself to form millions of copies.
In PCR a single strand of DNA generates
another strand by DNA polmerase using a
short complmentary DNA fragment this is
done using a primer which acts as an
initiating template.

89

Hypersensitivity is defined as a state of

exaggerated immune response to an
antigen.
Type 1: anaphylactic reaction.
Type 2: cytotoxic reaction.
Type 3: Immune complex reaction.
Type 4: cell mediated reaction.

90

The clumping together of the bodys

own RBC by Ab produced against them.

91

This is used for detection of species

specific antigen in blood stains on cloth
or knife.
Potent antiserum is used for test which
should give positive result with 1:
20,000 diluted blood.

92

When these are relesaed in a

circulation as a result of injury they
may elicit immune response.

93