Antigen –antibody reaction (Part :01)

INTRODUCTION:
 “The interaction between antigen & antibody is called antigen– antibody reaction.
It is abbreviated as Ag-Ab reaction.”
 Antigens are those substance that stimulates the production of antibodies which,
when enter into the body it reacts specifically in a manner that are clearly visible.
 An antigen introduced into the body produces only specific antibodies and will react
with only those specific antigens.
 These antibodies appear in the serum and tissue fluids. All antibodies are considered
as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE.
 Antigen-antibody reaction is the basis of humoral immunity or antibody
mediated immune response
 These reactions are used for the detection of infectious disease causing agents and
also some non-specific Ag’s like enzymes.

 Antigen – antibody reactions are performed to determine the presence of either
the antigen or antibody. [serological tests (in vitro)].
 One of the two components has to be known. For e.g. with a known antigen,
such as influenza virus , a test can determine whether antibody to the virus is
present or not
 The reaction occurs mainly in 3 stages;
STAGE 1. The interaction between the antigen and antibody occurs without any
visible effects. It is a rapid reaction.
STAGE 2. The secondary stage leads to the visible events, such as precipitation,
agglutination, etc.
STAGE 3. The tertiary reaction follows the neutralization or destruction of
injurious antigens.

ANTIGEN – ANTIBODY REACTIONS
 Immune Complex.
 Specificity of Ag-Ab Reaction.
 Binding Sites of Ag-Ab Reaction.
 Binding Forces of Ag-Ab Reaction.
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1. IMMUNE COMPLEX:
 When antigen & antibody are brought together, the antibody binds with the
antigen to form a complex molecules called immune complex
or Ag- Ab complex.
Ag + Ab Ag – Ab COMPLEX
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2. SPECIFICITY OF Ag-Ab REACION:
 The ability of a population of antibody
molecules to react with only one antigen.
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3. Binding Sites of Ag-Ab Reaction.
 The part of antigen which combines with
antibody is called Epitope.
 An epitope, also known as antigenic
determinant, is the part of an antigen that
is recognized by the immune system,
specifically by antibodies or T cells.
 An antigen may contain 10 to 50 antigenic
determinants. Some time it may go up to
200.
 The part of an antibody that recognizes
the epitope is called a Paratope.
 Most of the antibodies are bivalent having
two binding sites.But the antibody IgM is
multivalent having 5 to 10 binding sites.
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Paratope

4. Binding Forces of Ag-Ab
Reaction.
 The binding between antigen and
antibody in Ag – Ab reaction is due
to three factors:
1. Closeness between antigen and
antibody: When antigen and
antibody are closely fit, the strength
of binding is great
2. Affinity of antibody: Antibody
affinity is the strength of the
reaction between a single antigenic
determinant (epitope) and a single
combining site on the antibody
(paratope).
3. Non – Covalent Bonds: The bonds
that hold the antigen to the antibody
combining site are all non-covalent
in nature.
 These include hydrogen bonds, Van
der Waals forces and hydrophobic
bonds.
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ANTIGEN – ANTIBODY REACTION:
1. AFFINITY:
 It refers to the intensity of the attraction
between antigen and antibody molecule.
2. AVIDITY:
 It is the strength of the bond after the
formation antigen and antibody complex.
3. CROSS REACTIVITY:
 The ability of an individual Ab
combining site to react with more
than one epitope
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TYPES OF ANTIGEN – ANTIBODY REACTION
The types of antigen – antibody reactions are:
 Precipitation Reaction.
 Agglutination Reaction.
 Complement Fixation.
 ELISA – Enzyme Linked ImmunoSorbent Assay.
 Immunofluorescence.

Antigen –antibody reaction (Part :01)

PRECIPITATION REACTION
“When a soluble antigen combines with its antibody in the presence of electrolytes
at a suitable temperature and PH, it forms an insoluble precipitate of the Ag-
Ab complex. The antibody causing precipitation is called Precipitin and the
reaction is called as precipitation reaction.”
 Precipitation occurs in 2 media:
a) Liquid.
-Ring test
-Slide test
-Tube test
b) Gel.
-Immunodiffusion.
-Electroimmunodiffusion.

a) Precipitation in Liquid:
 One of the easiest of serological tests.
 Soluble antigens interact with antibodies and form a lattice that
eventually develops into a visible precipitate.
 Occur best when antigen and antibody
are present in optimal proportions.
 Precipitin ring test is performed in a
small tube.
 Antibodies that aggregate soluble
antigens are called precipitins.

The precipitin curve shows
3 zones:
“Zone of
antibody
excess”
“Zone of
equivalence”
“Zone of
antigen
excess”
Clinical application:
This test is used for the standardization of toxins and toxoids.

b) Precipitation in Gel:
“Precipitation assays are performed in semi-solid media
such as agar where antibodies and antigens can diffuse
toward one another and form a visible line of
precipitation.”
 The most widely used standard precipitation methods are
Ouchterlony test (Double Diffusion) and Mancini test (Radial
Immunodiffusion).

in the Ab-containing semisolid medium
The region of equivalence
-> The area is
proportional to
the conc. of Ag.
Radial Immunodiffusion
(Mancini test)
•- In this method, antiserum
solution containing Ab is
incorporated in agar gel on a
slide or petri-plate.
•- The Ag is then applied to a well
cut into the gel.
•- When Ab already present in
the gel reacts with the Ag which
diffuses out of the well, a ring
of precipitation is formed
around the well.
•- The diameter of ring is directly
proportional to the
concentration of Ag.
•- RID has been used for the
quantitative estimation
of antibodies and antigens
in the serum.
Diagrammatic representation of radial & double immunodiffusion.
: Precipitation in gels yield visible precipitin lines;
no visible precipitate forms in regions of Ab or Ag excess.

AGGLUTINATION REACTION
“It is an antigen-antibody reaction, in which particulate antigen combines with its
antibody in the presence of electrolytes at a suitable temperature and PH.”
 Agglutination is the visible clumping of insoluble particles, whereas
precipitation involves the aggregation of soluble molecules.
 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.
 The agglutination is more sensitive than precipitation
for the detection of antibodies.
Clumping of particles

TYPES OF AGGLUTINATION REACTION
Slide
agglutination
test
Tube
agglutination
test
The
Antiglobulin
test
Passive
agglutination
test

SLIDE AGGLUTINATION
 This is a rapid method to determine the presence of agglutinating
antibodies.
 A uniform suspension of antigen is made in a drop of saline on a slide.
 To this a drop of antibody is added.
 A positive result is indicated by the clumping of together of particles.
 The particulate antigen include bacterial ,viruses ,RBC ,platelets
lymphocytes ,etc.
 When red blood called are agglutinated ,the reaction is called
Heamagglutination .
ABO blood types

SLIDE AGGLUTINATION
Applications:
 Used for blood grouping and cross matching.
 Used to identify bacterial strains.
Eg: Salmonella species
 Identification of
cultures of Shigella.

TUBE AGGLUTINATION TEST
 Tube agglutination test is a standard quantitative method for the measurement of antibodies.
 In this method fixed volume of particulate antigen suspension is added to an equal volume of
serial dilutions of an antibody (patient‘s serum) in test tubes.
 Control test tube is kept which has no antiserum.
 The tubes are incubated until visible agglutination is observed
 The tube showing highest agglutination is referred to as the “Agglutination titre”.
 Tube agglutination is employed for the
serological diagnosis of typhoid,
brucellosis and typhus fever.
Widal test is used for the estimation
of typhoid fever. Agglutination Titre

THE ANTIGLOBULIN TEST
 There are two types of Coombs test:
Coombs test
Direct Coombs test Indirect Coombs test
 Direct Coombs test: sensitization of the erythrocytes with incomplete
antibodies takes place in vivo
 Indirect Coombs test: sensitization of the erythrocytes with incomplete
antibodies takes place in vitro

Recipient
Donor
Anti-human IgG
(COOMB’S REAGENT)
Anti-human IgG
(COOMB’S REAGENT)

APPLICATIONS:
 This test is performed to detect presence of Rh-Antibodies or other
antibodies in patients serum in case of the following:
1. To check whether an Rh-negative women (married to Rh-positive
husband) has developed Rh-antibodies.
2. Anti D may be produced in the blood of any
Rh-negative person by exposure to D Ag by-
* Transfusion of Rh-positive blood
* Pregnancy, if infant is Rh-positive (if father
is Rh Positive).
* Abortion of Rh-positive fetus.

PASSIVE AGGLUTINATION TEST
 A precipitation reaction can be converted into agglutination test by
attaching the soluble antigens to the surface of carrier particles
like latex particles and red blood cells.
 Such a type of test is called as “passive agglutination test”.
 This test is used for the diagnosis of Rheumatoid Arthritis (Rose-
Waller test).

Antigen –antibody reaction (Part :01)

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Antigen –antibody reaction (Part :01)