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Agglutination Reactions PDF
Agglutination Reactions PDF
Agglutination- Reactions
AGGLUTINATION REACTIONS
Agglutination is the visible aggregation of particles caused by combination with specific antibody.
Reaction takes place on the surface of the particle, antigen must be exposed and able to bind
with antibody
A two-step process, involving sensitization or initial binding followed by lattice formation, or
formation of large aggregates.
RBCs, Bacterial cells, inert carriers (latex particles) – multiple antigenic or determinant sites
Gruber and Durham (1896) – first to report the ability of antibody to clump cells, based on observations of
agglutination of bacterial cells by serum.
Widal and Sicard – detection of antibodies occurring in typhoid fever, brucellosis and tularemia.
Sensitization - antigen–antibody combination through single antigenic determinants on the particle surface
and is followed by the law of mass action and is rapid and reversible.
The affinity and avidity of an individual antibody determine how much antibody remains
attached.
The class of immunoglobulin (IgM is more efficient than IgG)
If epitopes are sparse or if they are obscured by other surface molecules, they are less likely to
interact with antibody
Lattice Formation - sum of interactions between antibody and multiple antigenic determinants on a particle, is
dependent on environmental conditions and the relative concentrations of antigen and antibody
Bordet - governed by physicochemical factors such as the milieu’s ionic strength, pH, and temperature
Antibody must be able to bridge the gap between cells in such a way that one molecule can bind to a
site on each of two different cells
1. DIRECT AGGLUTINATION
2. PASSIVE AGGLUTINATION
3. REVERSE PASSIVE AGGLUTINATION
4. AGGLUTINATION INHIBITION
5. COAGGLUTINATION
Passive/Indirect Agglutination - employs particles that are coated with antigens not normally found on their
surfaces.
Carrier particles:
RBCs – possibility of cross-reactivity (heterophile antibody)
Latex – inexepensive, relatively stable, not subject to cross-reactivity
Gelatin
Silicates
Use to detect:
Rheumatoid Factor
Antinuclear antibody
ASO
Abs to Trichinella spiralis
Abs to Treponema pallidum
Abs to CMV, Rubella, Varicella-zoster, and HIV-1/2
There is always a risk of non-specific agglutination caused by the presence of other IgM antibodies
Reverse Passive Agglutination - antibody rather than antigen is attached to a carrier particle.
The antibody must still be reactive and is joined in such a manner that the active sites are facing
outward.
Adsorption may be spontaneous, or it may require some of the same manipulation as is used for
antigen attachment.
Agglutination Inhibition - reactions are 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.
Haptens that are complexed to proteins; the hapten–protein conjugate is then attached to a
carrier particle.
The patient sample is first reacted with a limited amount of reagent antibody that is specific for the
hapten being tested. Indicator particles that contain the same hapten one wishes to measure in the
patient are then added.
If the patient sample has no free hapten, the reagent antibody is able to combine with the carrier
particles and produce a visible agglutination. – agglutination is a negative reaction, indicating that
the patient did not have sufficient hapten to inhibit the secondary reaction
Either antigen or antibody can be attached to the particles.
The sensitivity of the reaction is governed by the avidity of the antibody itself. It can be a highly
sensitive assay capable of detecting small quantities of antigen.
Hemagglutination Inhibition – reactions are the same principle, except RBCs are the indicator particles.
Coagglutination - systems using bacteria as the inert particles to which antibody is attached.
Staphylococcus aureus is most frequently used, because it has a protein on its outer surface, called
protein A, which naturally adsorbs the fragment crystallizable (FC) portion of antibody molecules.
These particles exhibit greater stability than latex particles and are more refractory to changes in
ionic strength.
Antiglobulin-Mediated Agglutination
AHG (Coomb’s 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.
DAT – used to demonstrate in-vivo attachment of antibody or complement to an individual’s 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.
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 a specific blood group antigens.