The Journal of Cell Biology, Vol 90, 249-253, Copyright © 1981 by The Rockefeller University Press
Detection and characterization of monoclonal antibodies to platelet membrane proteins
PJ Newman, RA Kahn and A Hines
We have devised a solid-phase radioimmunoassay for the detection and
characterization of monoclonal antibodies directed against platelet surface
antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100,
were covalently coupled to cyanogen bromide (CNBr)- activated filter paper
disks that were than used as the support in antibody binding assays. SDS
PAGE of solubilized membrane proteins taken immediately before and after
incubation with activated disks indicated that representative amounts of
each membrane protein were bound to the disks. Either monoclonal or
heterologous anti-platelet antibody could be detected on disks that had
been prepared using as little as 50 micrograms of membrane protein per 100
disks. For the detection of antibody, disks were incubated with test sera
for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and
the amount of bound radioactivity was determined. The sensitivity of the
disk assay in detecting monoclonal antibodies was far greater than that of
a corresponding radioimmunoassay that used whole platelets as the solid
phase. By linking other proteins such as fibrinogen or anti-mouse subclass
specific antisera to CNBr-activated disks, the method was adapted for
antibody characterization. The sensitivity and ease with which the assay
can be performed make this technique most suitable for screening and
characterizing monoclonal antibodies.
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