Agglutination is the clumping of particles that have antigens on their surface, such as erythrocytes, by antibody molecules that form bridges between the antigenic determinants. This is the end point for most tests involving erythrocyte antigens. Agglutination is influenced by a number of factors and is believed to occur in two stages, sensitization and lattice formation.

Sensitization

The first phase of agglutination, sensitization, represents the physical attachment of antibody molecules to antigens on the erythrocyte membrane. The combination of antigen and antibody is a reversible chemical reaction. Altering the physical conditions can result in the release of antibody from the antigen-binding site. When physical conditions are purposely manipulated to break the antigen-antibody complex, with subsequent release of the antibody into the surrounding medium, the procedure is referred to as an elution.

The amount of antibody that will react is affected by the equilibrium constant, or affinity constant, of the antibody. In most cases, the higher the equilibrium constant, the higher is the rate of association and the slower the rate of dissociation of antibody molecules. The degree of association between antigen and antibody is affected by a variety of factors and can be altered in some cases in vitro by altering some of the factors that influence antigen-antibody association, including the following:

 • Particle charge

 • Electrolyte concentration and viscosity

• Antibody type

• Antigen-to-antibody ratio

• Antigenic determinants

 • Physical conditions (e.g., pH, temperature, duration of incubation)

Particle Charge. Inert particles such as latex, RBCs, and bacteria have a net negative surface charge called the zeta potential (Fig. 1). The concentration of salt in the reaction medium has an effect on antibody uptake by the membrane bound erythrocyte antigens. Sodium (Na⁺) and chloride (Cl⁻) ions in a solution have a shielding effect. These ions cluster around and partially neutralize the opposite charges on antigen and antibody molecules, which hinders antibody-antigen association. By reducing the ionic strength of a reaction medium (e.g., using low ionic strength saline [LISS]), antibody uptake is enhanced. Charges can be overcome by centrifugation, addition of charged molecules (e.g., albumin, LISS), or enzyme pretreatment to permit the cross-linking that results in agglutination (Table 1).

Fig1. Zeta potential. Difference in electrostatic potential  between net charge at cell membrane and charge at surface of shear.  (From Turgeon ML: Fundamentals of immunohematology, ed 2, Baltimore, 1995, Williams & Wilkins.)

Table1. Techniques to Reduce Zeta Potential

Antibody Type. Immunoglobulin M (IgM) antibodies are more efficient at agglutination because their large size and multivalency permit more effective bridging of the space between cells caused by zeta potential. IgG antibodies are too small to overcome electrostatic forces between cells. The use of AHG forms cross-links between antibody molecules that have bound to the surface of RBCs. This promotes this formation of agglutination and allows for visual observation of an antigen antibody reaction.

Antigen-Antibody Ratio. Under conditions of antibody excess, there is a surplus of molecular antigen-combining sites not bound to antigenic determinants. Precipitation reactions depend on a zone of equivalence, the zone in which optimum precipitation occurs, because the number of multivalent sites of antigens and antibodies are approximately equal. For a precipitation reaction to be detectable, the reaction must occur in the zone of equivalence. In this zone, each antibody or antigen binds to more than one antigen or antibody, respectively, forming a stable lattice or network. This lattice hypothesis is based on the assumptions that each antibody molecule must have at least two binding sites and that an anti gen must be multivalent.

On either side of the zone of equivalence, precipitation is prevented because of an excess of antigen or antibody. If excessive antibody concentration is present, the phenomenon known as the prozone phenomenon occurs, which can result in a false-negative reaction. In this case, antigen combines with only one or two antibody molecules and no cross-linkages are formed. This phenomenon can be overcome by serially diluting the antibody-containing serum until optimum amounts of antigen and antibody are present in the test system.

If an excess of antigen occurs, the postzone phenomenon occurs, in which small aggregates (clumps) are surrounded by excess antigen and no lattice formation is established. Excess antigen can block the presence of a small amount of antibody. To correct the postzone phenomenon, a repeat blood specimen should be collected 1 or more weeks later. If an active antibody reaction is occurring in vivo, the titer of antibody will increase and should be detectable. Repeated negative results generally suggest that the patient has the specific antibody being tested for by the procedure.

Antigenic Determinants. The placement and number of antigenic determinants both affect agglutination. For example, the A blood group antigen has more than 1.5 million sites/RBC, whereas the Kell blood group antigen has about 3500 to 6000 sites/RBC. If the number of antigenic sites is small or if the antigenic sites are buried deeply in the cell membranes, antibodies will be unable physically to contact antigenic sites.

Steric hindrance is an important physiochemical effect that influences antibody uptake by cell surface antigens. If dissimilar antibodies with approximately the same binding constant are directed against antigenic determinants located close to each other, the antibodies will compete for space in reaching their specific receptor sites. The effect of this competition can be mutual blocking, or steric hindrance, and neither antibody type will be bound to its respective antigenic determinant. Steric hindrance can occur whenever there is a con formational change in the relationship of an antigenic receptor site to the outside surface. In addition to antibody competition, competition with bound complement, other protein molecules, or the action of agents that interfere with the structural integrity of the cell surface can produce steric hindrance.

pH. The pH of the medium used for testing should be near physiologic conditions, or an optimum pH of 6.5 to 7.5. At a neutral pH, high electrolyte concentrations act to neutralize the net negative charge of particles.

Temperature and Length of Incubation. The optimum temperature needed to reach equilibrium in an antibody-antigen reaction differs for different antibodies. IgM antibodies are cold-reacting (thermal range, 4° C to 22° C [39° F to 72° F]), and IgG antibodies are warm-reacting, with an optimum temperature of reaction at 37° C (98.6° F).

The duration of incubation required to achieve maximum results depends on the rate of association and dissociation of each specific antibody. In laboratory testing, incubation times range from 15 to 60 minutes. The optimum time of incubation varies, depending on the class of immunoglobulin and how tightly an antibody attaches to its specific antigen.

Lattice Formation

Lattice formation, or the establishment of cross-links between sensitized particles (e.g., erythrocytes) and antibodies, resulting in aggregation, is a much slower process than the sensitization phase. The formation of chemical bonds and resultant lattice formation depend on the ability of a cell with attached antibody on its surface to come close enough to another cell to permit the antibody molecules to bridge the gap and combine with the antigen receptor site on the second cell. As antigens and antibodies combine, a multimolecular lattice increases in size until it precipitates out of solution as a solid particle. Cross-linking is influenced by factors such as the zeta potential.

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مواضيع ذات صلة

Antibody Structure and Function

B Cell Receptor for Antigen

Antigen Processing and Presentation

Methods of Enhancing Agglutination

The Major Histocompatibility Complex

Cellular Basis of the Adaptive Immune Response

Mechanisms of Innate Immunity

Overview of Acute-Phase Proteins

C-Reactive Protein

Hematopoietic Stimulators

The Complement System: Diagnostic Evaluation

Biological Functions of Complement Proteins