Ag-Ab CoM 21.pptantigen antibody interaction

Ag-An interaction

1. Antigen-Antibody Interaction
(SEROLOGY)

2. 
The antigens and the antibodies combine specifically with
each other. This interaction between them is called Antigen-
Antibody reaction.
These form the basis for humoral immunity or antibody
mediated immunity.
These reactions form the basis for detection of infectious
disease causing agents.
When Ag – Ab reactions occur in vitro, they are known as
serological reactions.
INTRODUCTION:

3. Three Distinct Phases of Antigen/Antibody
Reactions
Primary Phenomenon – Sensitization
*involves attachment of antibody to it’s specific antigen.
 

* Rapid and reversible reaction.
 

*This step affected by antibody affinity and avidity which will determine
how much antibody remains attached.
Secondary Phenomenon – Lattice formation (in vitro)

Tertiary Phenomenon – Detected by affect on tissues or cells.(in vivo)
*Phagocytosis
*Antibody-dependent cellular cytotoxicity (ADCC) and
*Release of inflammatory mediators
*Complement activation,
*Opsonization of target cells
* Neutralization.

4. Affinity vs. Avidity
Affinity
Measure of the binding strength between an antigenic determinant
(epitope) and an antibody combining site.
Is the strength of the reaction between a single antigenic determinant and
a single combining site on the antibody.
Avidity
• It is the strength of the bond after the formation of Ag-Ab
complexes.
• It is used to denote the overall capacity of antibodies to
combine with the multivalent antigen.
• A multivalent Ag has many types of antigenic determinants.
• When injected into the blood, each antigenic determinant
stimulates the production of a particular antibody.

6. Ag-Ab reaction bonds:

•Hydrogen
•Ionic
•Hydrophobic
interactions
•Van der Waals forces

7. Secondary Phenomenon
To detect Immune complex in vitro , numerous
types of serologic tests differ in their speed and
sensitivity.
Some are strictly qualitative , others are
quantitative.
These Tests include:
1.Precipitation
2.Agglutination
3.Neutralization
4.Complement fixation
5.5.ImmunofluorescenceImmunofluorescence
6.6.Radioimmunoassay (RIA)Radioimmunoassay (RIA)
7.7.Enzyme-Linked Immuno sorbent Assay (ELISA)Enzyme-Linked Immuno sorbent Assay (ELISA)

8. Precipitation Reaction:
When a soluble Ag combines with its Ab in the
presence of an electrolyte (NaCl) at a particular
temperature and pH, it forms an insoluble precipitate
of Ag-Ab complex. The Ab causing precipitation is
called Precipitin and the reaction is called as
precipitation reaction.
Antibodies Antigens Ag-Ab complex

9.  Function of precipitation reaction: Precipitation occurs
in two media:
Precipitation in Liquid:
Antigen – Antibody reaction perform by placing a constant
amount of antibody in a series of tubes and adding increased
amount of antigen. Antigen – Antibody reacts together
resulting in precipitation.
Plotting the amount of precipitate against increasing antigen
concentration yeilds a precipitation curve.

10. Precipitation in gel:
I- Simple Immunodiffusion (ID)
a)Double ID (Ouchterlony)
b)Single Radial ID (RID) (Mancini)
II- Electro-Immnodiffusion
a)Immunoelectrophoresis (IEP)
b)Rocket Electroimmunodiffusion (EID)
c)Counterimmunoelectrophoresis (CIEP)
III-Measurement of Precipitation by Light
a)Turbidimetry
b)Nephelometry

11. If Ag or Ab preparations
are complex, multiple
bands form. These are:
*Radial Immunodiffusion
(Mancini)
*Doublediffusion methods
Precipitation reactions in gels

13. Cross Reaction:
An antiserum raised against an Ag, can also react with a
similar Ag of another type. This is called cross reaction
and the Ag which produces the cross reaction is called
Cross reactive Ag. But the strength of Ab raised against its
own Ag is strong.
Examples:
*The antiserum raised against S. pyogenes cross react with
heart valve causing rheumatic fever.
*The antiserum raised against Pneumococcal
polysaccharides will react with E.coli, blood group A and
collagen Ag’s.

14. Antigen Added

15. Agglutination Reaction:
• When a particulate Ag is mixed with its Ab’s in the
presence of electrolytes at a suitable temperature and pH,
the particles are clumped or agglutinated.
• The Ab of the serum causes the cellular Ag’s to form
clumps and these are called Agglutinins.
• The particulate antigens that are aggregated are termed
Agglutinogens.

16. Slide agglutination: This is a rapid method to
determine the presence of agglutinating antibodies

17. Tube agglutination:
This is a standard method for quantitative estimation of Ab.
The serum containing Ab is diluted serially with saline in
several small test tubes, to which a constant volume of Ag
suspension is added.
A control tube is kept which has no antiserum. The tubes are
incubated until The tube showing highest agglutination is
referred to as the Titer.
visible agglutination is observed.
Ex: typhoid (Widal), brucellosis (Rose Bengal).

18. Tube Agglutination
In this test Ab content of the patient’s serum, is
measured by adding a constant amount of antigen
(Solmonella typhi) to the serially diluted serum.

19. Passive agglutination test:
It is similar to haemagglutination test but the physical nature
of the reaction is altered.
The Ag is coated on the surface of a carrier particle and
thereby helps to convert a precipitation reaction into an
agglutination reaction making the reaction more sensitive.
The carrier particles used can be RBC, latex particles or
bentonite. Some times RBC coated with polystyrene (tanned
RBC) can be used.
When patients serum is mixed with these, it leads to
agglutination. This test is used for the diagnosis of
Rheumatoid Arthritis (RF)

20. Passive Agglutination
Carrier
Particle
Soluble
Antigen
Coated
Particle
Coated ParticleAntibody Agglutination

21. Haemagglutination test

22. Agglutination InhibitionAgglutination Inhibition
TestsTests
Pregnancy
Testing
-classic example of agglutination inhibition
Human
chorionic
gonadotropin
(hCG)
Appears in serum
and urine early in
pregnancy

23. Complement Fixation
Principle. When antigen and antibodies of the IgM
or the IgG classes are mixed, complement is “fixed”
to the antigen-antibody aggregate. If this occurs on
the surface of a red blood cell, the complement
cascade will be activated and hemolysis will occur
Applications: widely applicable to the detection of
antibodies to almost any antigen.
Wassermann test for syphilis
Antibodies to Mycoplasma pneumoniae.

25. PrinciplePrinciple
use of enzyme-labeled immunoglobulin to
detect antigens or antibodies
signals are developed by the action of
hydrolyzing enzyme on chromogenic substrate
optical density measured by micro-plate reader
Examples
Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)
Cytokines level
ANA, ENA
Enzyme-linked Immuno-Enzyme-linked Immuno-
Sorbant assay (ELISA) Sorbant assay (ELISA)

26. ELISA
2626
Antibody
R
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s
p
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Micro-plate reader
96-well micro-plate
Positive result

27. 1- Non-competitive 1- Non-competitive
must remove excess/unbound Ag or Ab before
every step of reactions
Direct ELISA
Indirect ELISA
Sandwich ELISA
Ab Capture ELISA (similar to sandwich ELISA but in 1
st
step,
anti-Ig (M or G) is coated on the plate
Then antibodies in patient serum are allowed to capture in next
step
Types of ELISA used in the detection of Ag or AbTypes of ELISA used in the detection of Ag or Ab
2-Competitive ELISA2-Competitive ELISA

28. Labeling
techniqueTypes of ELISA (Ag Abs tests)Types of ELISA (Ag Abs tests)

29. Types of ELISA

31. Immunofluorescence:
•Fluorescence is the property of absorbing light rays of one
particular wavelength and emitting rays with a different wave
length.
•Dyes that are commonly used include:
Fluorescein, an organic dye that is the most widely used label for
immunofluorescence procedures, absorbs blue light (490 nm) and
emits an intense yellow-green fluorescence (517 nm).
Phycoerythrin is an efficient absorber of light (~30-fold greater
than fluorescein) and a brilliant emitter of red fluorescence,
stimulating its wide use as a label for immunofluorescence.
•

33. Principle
Use fluorescein isothiocyanate
labeled-immunoglobulin to detect
antigens or antibodies according to
test systems
Requires a fluorescent microscope
Examples
Herpes virus IgM
Dengue virus
Rabies virus
typhus
ANA patterns
Immuno-fluorescence
Cell infected with Dengue
virus
V.
Cholerae