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Agglutination- Reactions

Medical Technology (Our Lady of Fatima University)

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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

Enhancement of Lattice Formation


 LISS – decreasing the buffer’s ionic strength
 Albumin – 5 to 30% helps to neutralize the surface charge and allows red cells to approach each
other more closely
 Increase viscosity using enzymes, agitating centrifuging, altering temperature or the pH
 PEG and Dextran – reduce the water hydration around cells and allow them to come into closer
proximity for antibody to join together
 Bromelin, Papain, Trypsin and Ficin – reduces the surface charge on the RBCs through cleaving
of chemical groups and decreasing hydration.
 Ficin – cleaves sialoglycoproteins from the RBCs surface and may change the external
configuration of the membrane to reveal more antigenic determinant sites.
 IgGs reacts best at 30 to 37 degree Celsius, IgM reacts best between 4 to 27 degree Celsius
 pH – optimal 6.5 – 7.5

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TYPES OF AGGLUTINATION REACTIONS

1. DIRECT AGGLUTINATION
2. PASSIVE AGGLUTINATION
3. REVERSE PASSIVE AGGLUTINATION
4. AGGLUTINATION INHIBITION
5. COAGGLUTINATION

Direct Agglutination - occurs when antigens are found naturally on a particle.


 patient serum is diluted into a series of tubes or wells on a slide and reacted with bacterial antigens
specific for the suspected disease
 Used in diagnosis of diseases for which the bacterial agents are extremely difficult to cultivate.
 fourfold increase in antibody titer over time when paired dilutions of serum samples are tested
with any of these antigens

Hemagglutination - agglutination reaction involves red blood cells


Examples: ABO typing

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.

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 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

1. DIRECT ANTI-GLOBULIN TEST


2. INDIRECT ANTI-GLOBULIN TEST

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.

 All reactions are run at 37°C to detect clinically significant antibodies.


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

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