AGGLUTINATIO

N Presented By : Garima Dutta

TABLE OF CONTENTS

Types of
01 Agglutination 02 Applications of
Agglutination

03 Advantages 04 Limitations
● Visible clumping of cell or cellular antigen by binding of
antibody

● Agglutinate

● In presence of electrolyte at optimum pH of 7.4 and

temperature of 37°C

● Very high concentration of antigen gives negative result - post-

zone effect

● Very high concentration of antibody also inhibits

● Very high concentration of antibody also inhibits agglutination
reaction. - pro-zone effect

● Pro-zone effect also occurs due to high concentration of

incomplete or blocking antibody (eg. IgA).

● Blocking antibody lack flexibility and fail to agglutinate cell.

● Only when antigen and antibody are in appropriate

concentration.
 Agglutination is the development of antigen–antibody
complexes in the form of particle clumps, due to the
interaction between the insoluble form of antigens and its
soluble and specific antibodies
 0
1
TYPES
Of Agglutination
 01 02 03

Active/Direct Passive
Hemagglutination
Agglutination Agglutination
 1. Active Agglutination
• Where antigens are found naturally on a particle - direct
agglutination
• In active agglutination, direct agglutination of particulate
antigen with specific antibody
• Uses whole pathogens as a source of antigen
• Measures the antibody level produced by a host infected with
that pathogen
i. Slide/Tile Agglutination:

 Basic type - performed on a slide

 Identification of bacterial types
 Suspension of unknown antigen is kept on
slide and a drop of standardized antiserum is
added or vice versa
 A positive reaction - formation of visible
clumps
 E.g. Widal test, RPR test.
ii. Tube Agglutination:

 Performed in tube and standard quantitative

technique for determination of antibody titre

 Serum is diluted in a series of tubes and standard

antigen suspensions are added

 After incubation, antigen-antibody reaction is

indicated
iii. Heterophile Agglutination:

● depends on demonstration of
heterophilic antibodies in serum
present in certain bacterial infections
iv. Antiglobulin (Coombs) Test:

● Devised by Coombs, Mourant, and Race

● For detection of incomplete anti-Rh antibodies that do not
agglutinate Rh+ erythrocytes in saline
● Serum containing incomplete anti-Rh antibodies - mixed
with Rh+ erythrocytes in saline - incomplete antibody
antiglobulin coats the surface of erythrocytes, does not
cause any agglutination
● Such erythrocytes treated with antiglobulin or Coombs
serum – agglutination
● direct as well as indirect
 DIRECT
The sensitization of RBCs with incomplete
antibodies takes place in vivo.
Cell-bound antibodies can be detected
Antiserum against human immunoglobulin
INDIRECT
The sensitization of RBCs with incomplete
antibodies - in vitro.
Patient’s serum is mixed with normal red
cells and antiserum to human
immunoglobulin Agglutination occurs if
antibodies are present in serum.
 2. Passive Agglutination
• Carrier particles that are coated with soluble antigens
• Either antibody or antigen is attached to certain inert carrier - particles or cells gets
agglutinated.
• Latex particles, Carbon particles, Bantonite etc - inert carriers.
• Antigens coated in latex particles used in ASO test
• Antibody instead of antigens is adsorbed on the carrier particle for detection of antigens
- reverse passive agglutination.
LATEX AGGLUTINATION:

● Latex particles as carrier of antigen or antibodies

● Many antibody or antigen molecules are bound
to latex beads - increases the number of antigen-
binding sites
● Antigen or antibody is present in a test specimen
- antigen antibody bind and form visible, cross-
linked aggregates
•
3. Hemaagglutination
RBCs - carrier particles
• RBCs of sheep, human, chick, etc. are commonly used
• RBCs are coated with antigen to detect antibodies in the serum - the test is
called indirect hemagglutination (IHA) test
• Uses erythrocytes as the biological carriers of bacterial antigens, and purified
polysaccharides or proteins for determining the presence of corresponding antibodies in
a specimen
• Antibodies are attached to the RBCs to detect microbial antigen - it is known
as reverse passive hemagglutination (RPHA)
VIRAL HEMAAGGLUTINATION:

● Viruses including influenza, mumps, and

measles - ability to agglutinate RBCs without
antigen–antibody reaction - viral
hemagglutination
● Can be inhibited by antibody specifically
directed against the virus - hemagglutination
inhibition
COAGGLUTINATION TEST:

● Cowan I strain of S. aureus - carrier particle to coat

antibodies
● Contains protein A, an anti-antibody, that combines
with the Fc portion of immunoglobulin, IgG, leaving
the Fab region free to react with the antigen
● In a positive test, protein A bearing S. aureus coated
with antibodies mixed with specific antigen -
Agglutination
 02
APPLICATIO
N
Of Agglutination
 • Blood grouping and cross matching

• IdentificationDetection
of unknown microbial culture, sero-typing

• Widal test,of antigen and antibody

• Weil-Felix test etc.

• The quantity of antibodies or antibody titer

• Diagnose disease due to a microorganism - difficult to detect or
grow directly in the clinical laboratory

• Detect antibodies to the three species of Brucella; Franciscella

tularensis, the cause of tularemia; and antibodies to Epstein-Barr
virus, the cause of mononucleosis

• Antigen of Cryptococcus neoformans

• Capsular antigens of Pneumococcus, Haemophilus influenzae and

Meningococcus

• Presence of beta-hemolytic Streptococcus

• Antistreptolysin O antibody

• Clostridium difficile toxins A and B, rotavirus, and Escherichia

coli 0157:H7
 03
ADVANTAGE
S
Of Agglutination
1
Require less time

High degree of analytic sensitivity

2
Can detect an enormous variety of

3 antibodies
 04
LIMITATION
S
Of Agglutination
1 Susceptibility to false-negative
reactions caused by the prozone
phenomenon

2 At best semi-quantitative
T H A N K
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