Comparison of serum anti-band 3 and anti-Gal antibody binding to
density-separated human red blood cells
MP Sorette, U Galili and MR Clark
Department of Laboratory Medicine, University of California, San Francisco
94143.
This study examines the quantitative relationship between two natural serum
antibodies, anti-band 3 and anti-alpha-galactosyl (anti-Gal), in their
capacity to bind to human red blood cell (RBC) populations separated on
density gradients. The question was approached in two ways. First, we
determined the extent of rebinding of affinity-purified human serum
antibodies to RBCs that had been stripped of in situ antibody. Second, we
eluted the in situ bound antibody at low pH from density-separated RBCs and
determined the proportion of total eluted antibody that bound specifically
to erythrocyte band 3 or to a Gal- alpha-(1,3)-Gal structure. Our results
show that high-density human RBCs bind increased amounts of both
antibodies. Anti-Gal rebinding was specific, because it was saturable and
occurred in the presence of serum IgG depleted of anti-Gal. Binding assays
using control natural autoantibodies directed against antigens not found on
the RBC surface showed that high-density RBCs also bind increased amounts
of these antibodies as compared with low-density RBCs. However, the extent
of this binding is substantially lower than that of anti-band 3 and anti-
Gal. Binding studies using the lectins Bandeiraea Simplicifolia (alpha-
galactosyl specific) and Arachis Hypogaea (peanut agglutinin, beta-
galactosyl specific) indicated that only the alpha-galactosyl sites are
exposed on high density RBCs, and not the beta-galactosyl structure
characteristic of T antigen. Antibody that is eluted at low pH from high
density RBCs contains a 5.0% to 18.0% component that binds to band 3
protein, and a 9.1% to 39.0% component that recognizes the alpha-
galactosyl structure. Together, the two antibodies appear to constitute an
average of 35% (range 17.2% to 57.4%) of the in situ bound antibody from
high-density human RBCs.
Volume 77,
Issue 3,
pp. 628-636,
02/01/1991
Copyright © 1991 by The American Society of Hematology
