Use of a sensitive bioimmunoabsorbent assay to isolate and characterize
monoclonal antibodies to biologically active human erythropoietin
AW Wognum, PM Lansdorp, CJ Eaves and G Krystal
Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada.
At present, one of the most sensitive assays for human erythropoietin (Ep)
is a bioassay that measures the Ep-dependent proliferation of spleen cells
from phenylhydrazine-treated mice after 24 hours in culture. We describe
how this assay can be used as the basis of a very sensitive method for
detecting mouse antibodies to biologically active human Ep. In this
procedure, microtiter wells are first coated with goat anti-mouse Ig
antibody, then treated with mouse antibodies (serum or hybridoma culture
supernatants), and finally incubated with a fixed amount of pure human Ep.
Specific binding of anti-Ep antibodies is detected by adding spleen cells
from phenylhydrazine-treated mice to the wells and measuring the ability of
the cells to incorporate 3H- thymidine 24 hours later. This
bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies
in sera from mice immunized with either pure human urinary Ep or a
synthetic dodecapeptide corresponding to the aminoterminal region of Ep and
in the culture supernatants from three of eight stable anti-Ep
antibody-producing hybridoma cell lines that we have isolated. The three
monoclonal antibodies showed similar reactivities in the BISA, but showed
different affinities for Ep, with Kd values of approximately 0.7, 8, and
240 nmol/L, respectively. Further studies showed that all antibodies were
capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a
radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed
to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our
results suggest that the BISA strategy may be an important complement to
conventional RIA and ELISA techniques for identification of monoclonal
antibodies specific for biologically active growth factors.
Volume 71,
Issue 6,
pp. 1731-1737,
06/01/1988
Copyright © 1988 by The American Society of Hematology
