Many antigen–antibody reactions occur in vitro but the reaction
commonly encountered in a blood is agglutination.
Agglutination is the clumping of those particles with antigens on
their surface, such as erythrocytes, by antibody molecules that form bridges
between the antigenic determinants.
Two stages of agglutination and the factor
that influence each:
1.
Sensitization or adsorption
Sensitization represents the
physical attachment of antibody molecules to antigens on the erythrocyte
membrane. The amount of antibody that will react is affected by the equilibrium
constant, affinity constant of the antibody. In most cases, the higher
the equilibrium constant, the higher the rate of association and the slower the
rate of dissociation of antibody molecules.
a.
Antigen–antibody ratio
Under conditions of antibody
excess, a surplus of molecular antigen–combining sites which are not bound to
antigenic determinants exists. The outcome of excessive antibody concentration
is known as the prozone phenomenon which can produce
false–negative reactions. This phenomenon can be overcome by serially
diluting the antibody–containing serum until optimum amounts of antigen
and antibody are present in test system.
b.
pH
A pH of 5.5 – 8.5 (average of
7.0) is used for routine laboratory testing.
c.
Temperature and length of
incubation
IgM (19S) type of antibodies are
cold reacting (thermal range: 4–22oC) and IgG (7S) antibodies are
warm reacting with an optimum temperature reaction of 37oC.
In laboratory testing, incubation
time range from 15–60 minutes. The optimum time of incubation varies according
to the class of immunoglobulin and how tightly an antibody attaches to specific
antigen.
d.
Ionic strength
Ionic strength is a measure of
intensity of the electric field due to ions in solutions. Sodium (Na+) and
Chloride (Cl–) in a solution have a shielding effect. These ions
cluster around and partially neutralize the opposite charges on antigen and
antibody molecules which hinder the association of antibody with antigen.
Reducing or lowering the ionic strength of a reaction medium with agents such
as low ionic strength saline (LISS) or polybrene enhances antibody uptake.
e.
Steric hindrance
This is the mutual blocking of
dissimilar antibodies with the same binding constant and directed against
antigenic determinants located in close proximity to each other on cell’s
surface. Steric hindrance can occur whenever a conformation change in the
relationship of an antigenic receptor site to the outside surface occurs. In
addition to antibody competition, competition with bound complement, other
protein molecule or the action of agents that interfere with the structural
integrity of the cell surface can produce steric hindrance.
2.
Lattice formation
Lattice formation or the
establishment of cross links between sensitized particles such as erythrocytes
and antibodies resulting in aggregation (clumping), 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.
a.
Zeta potential
Because of like charges repel one another; erythrocytes in suspension remain separated from each other. The actual distance of separation is governed by the effective net surface charge density. When erythrocytes are suspended in electrolyte solutions such as sodium chloride, the ions in a suspending solution arrange themselves about the surface of the cell and become more diffuse as the distance from the cell surfaces increases. As an erythrocyte floats in an electrolyte solution, some ions remain with it. The diffuse double layer surrounding the cell is called the ionic cloud and the outer edge of this layer is referred to as the surface of shear.
A difference in electrostatic
potential exists between the net charge of the cell membrane and the charge at the
surface of shear. This electrostatic potential is referred to as zeta
potential. Zeta potential is believed to depend on the actual net
surface charge density at the surface of the cell membrane and the
dielectric constant (the measure of the electrical conductivity of a suspending
medium) of the surrounding medium.
b.
Antibody type
Immunoglobulins are relatively
positively charged and following sensitization or coating by particles, they
reduce the zeta potential. Antibodies can bridge charged particles by extending
beyond the effective rage of the zeta potential which results in the
erythrocytes closely approaching each other, binding together and
agglutinating.
IgM antibodies are more efficient
in producing in vitro agglutination in 0.85% saline than IgG or IgA.
c.
Antigen dosage
The term “double dose”
is sometimes used to depict that homozygous genotypic expression of an antigen
(such as “cc), in comparison to the heterozygous genotypic expression of an
antigen (such “Cc”), which is referred to as a single dose. In some
blood group system, the presence of a homozygous genotype expresses itself with
more antigen than the heterozygous genotype. The consequence of possessing a
double dose of some blood group antigens (such as “c”) is that a greater
proportion of erythrocytes are agglutinated. This variation in strength of
agglutination is referred to as the dosage effect.
Method of enhancing agglutination
1.
Centrifugation
Centrifugation attempts to
overcome the problem of disturbance by subjecting sensitized cells to a high
gravitational force, which counteracts repulsive effect and physically forces
the cells together.
2.
Treatment with proteolytic enzyme
Treatment with a weak proteolytic enzyme can strip off some of the negative charges on the cell membrane by
removing surface sialic acid (cleaving sialoglycoproteins from the
cell surface) residues, thereby reducing the surface charge of the cells,
lowering the zeta potential and permitting the cells to come closer together
for chemical link by specific antibody molecules. The risk of enzyme treatment
is that it destroys some blood group antigens such as M, N, and Duffy.
3.
Use of colloid media
The most frequently used
colloidal reagent is bovine albumin solution. This product is
manufactured from raw bovine serum. Processing results in a solution with a 22%
protein concentration and a pH of 7.2, specifically
designed for laboratory use. The solution additionally contains 0.1%
sodium azide as a preservative.
4.
Use of LISS media
The principle of low ionic
strength saline (LISS) is that, the rate of antibody association
increases as the ionic strength of the medium decreases. The use of LISS not
only increases the sensitivity of antibody detection compared to bovine albumin
but also allows a shortened period of incubation.
The most frequent disadvantage of
LISS technique is the increased number of non– specific or false positive
reactions observed. A more serious problem is the difficulty in detecting some
examples of anti–Kell using LISS.
5.
Use of PEG media
The addition of polyethylene glycol (PEG) to serum cell test mixtures has been reported to be more
effective in detecting weak antibodies than LISS and manual polybrene. In general,
polybrene glycol does not produce non–specific reactions.
6.
Use of polybrene media
The principle of this technique
is that the addition of macromolecules brings the red cell closer together to
facilitate strong cross–linking by cell–bound antibodies. It offers a benefit
of detecting clinically significant blood group alloantibodies failed to be
detected by saline–albumin technique.
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