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AGGLUTINATION REACTIONS Antibody must be able to bridge the gap

between cells in such a way that one molecule


Agglutination is the visible aggregation of particles can bind to a site on each of two different cells
caused by combination with specific antibody.
Enhancement of Lattice Formation
Reaction takes place on the surface of the LISS decreasing the buffers ionic strength
particle, antigen must be exposed and able
Albumin 5 to 30% helps to neutralize the
to bind with antibody
surface charge and allows red cells to
A two-step process, involving sensitization
approach each other more closely
or initial binding followed by lattice
formation, or formation of large aggregates. Increase viscosity using enzymes, agitating
RBCs, Bacterial cells, inert carriers (latex centrifuging, altering temperature or the Ph
particles) multiple antigenic or
PEG and Dextran reduce the water
determinant sites
hydration around cells and allow them to
come into closer proximity for antibody to
Gruber and Durham (1896) first to report the
join together
ability of antibody to clump cells, based on
observations of agglutination of bacterial cells Bromelin, Papain, Trypsin and Ficin
by serum. reduces the surface charge on the RBCs
through cleaving of chemical groups and
Widal and Sicard detection of antibodies decreasing hydration.
occurring in typhoid fever, brucellosis and Ficin cleaves sialoglycoproteins from the
tularemia. RBCs surface and may change the external
configuration of the membrane to reveal
Sensitization - antigenantibody combination more antigenic determinant sites.
through single antigenic determinants on the
particle surface and is followed by the law of IgGs reacts best at 30 to 370C, IgM reacts
mass action and is rapid and reversible. best between 4 to 270C
pH optimal 6.5 7.5
The affinity and avidity of an individual
antibody determine how much antibody
remains attached. TYPES OF AGGLUTINATION REACTIONS:
The class of immunoglobulin (IgM is more
1. DIRECT AGGLUTINATION
efficient than IgG)
2. PASSIVE AGGLUTINATION
If epitopes are sparse or if they are
3. REVERSE PASSIVE AGGLUTINATION
obscured by other surface molecules, they
4. AGGLUTINATION INHIBITION
are less likely to interact with antibody
5. COAGGLUTINATION
Lattice Formation - sum of interactions between
Direct Agglutination - occurs when antigens are
antibody and multiple antigenic determinants
found naturally on a particle.
on a particle, is dependent on environmental
conditions and the relative concentrations of Patient serum is diluted into a series of tubes
antigen and antibody or wells on a slide and reacted with bacterial
antigens specific for the suspected disease.
Bordet - governed by physicochemical factors such Used in diagnosis of diseases for which the
as the milieus ionic strength, pH, and bacterial agents are extremely difficult to
temperature cultivate.
Fourfold increase in antibody titer over time Haptens that are complexed to proteins; the
when paired dilutions of serum samples are haptenprotein conjugate is then attached
tested with any of these antigens to a carrier particle.
The patient sample is first reacted with a
Hemagglutination - agglutination reaction
limited amount of reagent antibody that is
involves red blood cells
specific for the hapten being tested.
Examples: ABO typing Indicator particles that contain the same
hapten one wishes to measure in the patient
Passive/Indirect Agglutination - employs particles are then added.
that are coated with antigens not normally found
If the patient sample has no free hapten, the
on their surfaces.
reagent antibody is able to combine with the
Carrier particles:
carrier particles and produce a visible
RBCs possibility of cross-reactivity
agglutination. agglutination is a negative
(heterophile antibody)
Latex inexepensive, relatively reaction, indicating that the patient did not
stable, not subject to cross-reactivity have sufficient hapten to inhibit the
Gelatin secondary reaction
Silicates
Either antigen or antibody can be attached
Use to detect: to the particles.
Rheumatoid Factor
The sensitivity of the reaction is governed by
Antinuclear antibody
the avidity of the antibody itself. It can be a
ASO
Abs to Trichinella spiralis highly sensitive assay capable of detecting
Abs to Treponema pallidum small quantities of antigen.
Abs to CMV, Rubella, Varicella-zoster,
and HIV-1/2 Hemagglutination Inhibition reactions are
the same principle, except RBCs are the
There is always a risk of non-specific indicator particles.
agglutination caused by the presence of
other IgM antibodies
Coagglutination - systems using bacteria as the
Reverse Passive Agglutination - antibody rather inert particles to which antibody is attached.
than antigen is attached to a carrier particle.
The antibody must still be reactive and is Staphylococcus aureus is most frequently
joined in such a manner that the active sites used, because it has a protein on its outer
are facing outward. surface, called protein A, which naturally
adsorbs the fragment crystallizable (FC)
Adsorption may be spontaneous, or it may
portion of antibody molecules.
require some of the same manipulation as
is used for antigen attachment. These particles exhibit greater stability than
latex particles and are more refractory to
Agglutination Inhibition - reactions are based on changes in ionic strength.
competition between particulate and soluble
antigens for limited antibody-combining sites, and
a lack of agglutination is an indicator of a positive
reaction.
Antiglobulin-Mediated Agglutination

1. DIRECT ANTI-GLOBULIN TEST


2. INDIRECT ANTI-GLOBULIN TEST

AHG (Coombs Test) - technique that detects non-


agglutinating antibody by means of coupling with a
second antibody.
Antibody will react with the FC portion of the
human antibody attached to red blood cells.
Agglutination
Takes place because the antihuman globulin
is able to bridge the distance between cells
that IgG alone cannot do.

DIRECT ANTI-GLOBULIN TEST (DAT)


- Used to demonstrate in-vivo attachment of
antibody or complement to an individuals
red blood cells.

Serves as an indicator of autoimmune


haemolytic anemia, HDN, Sensitization of
RBCs caused by the presence of drugs, or a
transfusion reaction.
RBCs are washed to remove any antibody
that is not specifically attached and then
cells are tested directly with antibody to IgG
or complement
A positive test indicates that an immune
reaction is taking place in that individual.

INDIRECT ANTI-GLOBULIN TEST (IAT)


- Used to determine the presence of a
particular antibody in a patient or it can be
used to type patient red blood cells for
specific blood group antigens.

All reactions are run at 37C to detect


clinically significant antibodies.
Used to check for the presence of clinically
significant alloantibodies in patient serum.