AGGLUTINATION REACTIONS

Agglutination

RBC
IgM Antibody RBC RBC

RBC
RBC RBC RBC

RBC
IgG Antibody

RBC RBC

RBC
Agglutination Reactions
Characteristics
 is the aggregation of particulate matter
due to combination with specific antibody.
 takes place on the surface of the particle
and thus antigen must be exposed and
able to bind with antibody
 Involve particulate antigens and antibodies
 Characteristics cont’d
 types of particles participating in such reactions
include: erythrocytes, bacterial cells, and inert
carrier such as latex particles.
 antigen consists of suspensions of
microorganisms, cells (rbc) or uniform particles
like latex
 Antigens may be:
 On a cell (direct agglutination)
 Attached to latex spheres (indirect or passive
agglutination)
 Characteristics cont’d
 Agglutination reaction is aided by
elevated temperature (37-56°C) and by:
 Movement which increases the contact
between antigen and antibody.
 Clear supernatant.
 Clumps aggregate and settle as large
visible clumps.
Agglutination Requirements

 Availability of stable cell or particle

suspension
 Presence of one or more antigens close to the
surface
 Knowledge that ‘incomplete’ or non-
agglutinating antibodies are not detectable
without modifications, e.g. antiglobulin
(Coomb’s) technic
Advantages

 Agglutination of insoluble native

antigens or antigen-coated particles
simple to read with or without the aid
of a microscope.
 Increased degree of sensitivity
 Great variety of detectable
substances
 Steps in Agglutination
1. Sensitization
 involves antigen-antibody combination through
single antigenic determinants on the particle
surface
 may be affected by the nature of the antibody
molecules, affinity and avidity of individual
antibody, and the nature of antigen-bearing
surface
Steps in Agglutination Reaction
2. Aggregative Stage
 represents the sum of interaction between
antibody and multiple antigenic
determinants on a particle
 dependent on environmental conditions as
well as the relative concentrations of antigen
and antibody
 it could be enhanced by LISS (Low ionic
strength saline), neutralizes surface charges
and facilitates agglutination (albumin 5 -
50%)
Uses of Agglutination Reactions

 Aid in the identification, by means of known

antisera (serum containing antibodies
speciftic for a given antigen), microorganisms
cultured from clinical specimens.

 Help estimate the titer of antibacterial

agglutinins in the serum of patients with
unknown diseases.
Types of Agglutination Reactions

 Direct Agglutination
 Passive Agglutination
 Reverse Passive Agglutination
 Agglutination inhibition
 Hemagglutination-inhibition
 Coagglutination/Conglutination
 Types of Agglutination Rxns.

 Direct agglutination - occurs when antigens

are found naturally on a particle (Serotyping of
Salmonella)
 e.g. identification of bacterial types
 O antigen (somatic) - compact, fine and
granular agglutination
 H antigen (flagellar) - form a loosely woven
network of clumped cells (coarse and floccular),
called snowflake agglutination
 hemagglutination (agglutination reaction
which involves rbc)
Direct Agglutination Reaction
Agglutination Reactions

 Passive agglutination - employs particles

that are coated with antigens not normally
found on their surfaces.
 Inert materials commonly used:
1. Bentonite 3. Colloidion
2. Latex particles 4. Colloidal charcoal
 Agglutination Reactions
 Reverse Passive agglutination - antibody rather
than antigen is attached to a carrier particle.
 Agglutination inhibition - based on competition
between particulate and soluble antigens for
limited antibody combining sites, and a lack of
agglutination is an indicator of a positive
reaction.
 Hemagglutination inhibition reactions -tests
the presence of antibodies to certain viruses,
such as rubella, mumps, measles, influenza, and
adenovirus
Hemagglutination
 Hemagglutination involves agglutination of RBCs.
 Viral hemagglutination inhibition tests for
antibodies by the antibodies' ability to prevent
viruses from agglutinating RBCs.

Figure 18.7
 Neutralization Reactions
 Eliminate the harmful effect of a virus or exotoxin

Figure 18.8b
 Agglutination Reactions
 Coagglutination/Conglutination - name given
to systems using bacteria as inert particles to
which antibody is attached (S. aureus).
 the Fc region of antibody attaches to protein A of
staphylococcal cell leaving the Fab region to
combine with the antigen
 killed staphylococcal cells coated with antibody can
be used to identify bacteria and detect soluble
extracellular bacterial antigens in specimens and
body fluids.
Reading Agglutination Reactions
Macroscopic
+4 One solid aggregate or clump of cells.
+3 Several large aggregates, clear background.
+2 Small to medium sized aggregates, clear background.
+1 Small aggregates, turbid reddish background.
± Weak granularity in cell suspension. A few macroscopic
agglutinates but numerous agglutinates microscopically
MF Any degree of agglutination in a sea of unagglutinated cells
Hem Hemolysis is interpreted as a positive reaction and may be graded
as complete or partial; hemolysis and agglutination may be
recorded on the same tube.
Ø No agglutination, smooth reddish background
Microscopic:
+ Positive - aggregates of at least 3-5 cells.
Ø Negative - no agglutination.
Agglutination Reactions
Microscopic Agglutination
Hemagglutination
Hemagglutination Inhibition
Agglutination Reactions
Latex
Agglutination