slipping plane. The effective charge (potential) of the RBC, called the zeta potential, is
determined at this plane and is responsible for the electrostatic repulsion between one
RBC and another.
(b) In the first stage of agglutination, reducing the ionic strength of the
medium decreases the electropositive clouds of cations surrounding the RBCs and
facilitates the interaction of electropositive lgG with the negatively charged RBC. This
absorption of antibody to the RBC reduces the electronegative charge of the RBC and
reduces the zeta potential, thereby accelerating the second stage. Experiments have
shown that the initial rate of association of
anti-RhO(D) with RhO(D)-positive RBCs is increased 1,000-fold by a reduction of ionic
strength from 0.17 to 0.03 (for example, instead of using 0.9 percent NaCI, 0.2 percent
NaCI in 7 percent glucose or 0.3M glycine is used as a RBC diluent).
(5) Antigen-antibody ratio. The rate at which antibody is bound to the cell,
and the quantity of antibody bound, depend on the concentration of cells and of
antibody. In general, an increase in sensitivity is obtained by increasing the amount of
antibody in relation to antigen. This is often achieved in the blood bank by using less
antigen in the form of weaker cell suspensions (for example, it is a more sensitive
technique to add one volume of two percent RBCs to two volumes of serum, than to add
one volume of ten percent RBCs to two volumes of serum). Some agglutination
reactions are weakened or even become negative in the presence of an excess of
antibody, the prozone reactions phenomenon. The optimal proportion of antigen to
commercial antiserum is usually determined by the manufacturer; the directions issued
with each antiserum should be followed.
c. The Second Stage (Agglutination).
(1) Once RBCs are sensitized, they may or may not directly agglutinate.
Blood group antibodies were characterized empirically before the immunoglobulin
classes were recognized. Those antibodies that could produce agglutination in a saline
medium were called "complete" antibodies or `bivalent" antibodies, and those that did
not were called "incomplete" antibodies or "univalent" antibodies. Current evidence
indicates that all antibodies are at least bivalent; that is, each molecule has at least two
antigen-combining sites. The term incomplete antibody is used to denote an antibody
that reacts with, but fails to cause visible agglutination of a saline suspension of RBCs
possessing the corresponding antigenic determinant; such antibodies tend to be of
(2) The failure of "incomplete" antibodies to produce agglutination in a saline
environment may be a result, in part, of location, number, and mobility of antigenic
determinants on the RBC surface, of the size and configuration of the antibody
molecule, and of the electrostatic forces involved.