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P388D1, macrophage-like cells secreted a plasmacytoma inhibitory
factor (PIF) that suppressed the growth of MOPC-315 and induced
apoptosis. The specific aim of this study was to prepared a panel
of monoclonal antibodies that neutralized the activity of PIF.
P388D1 culture supernatants were analyzed by a DEAE-Sephacel
chromatography, and the plasmacytoma inhibitory activity was
localized on two protein peaks designated as Peak 1 and Peak 2.
Both Peak 1 and Peak 2 were used to immunize Balb/c mices,
together and separately. The hybirdoma were prepared by fusing
spleen cells with myeloma and were screened using both
neutralization assay and ELISA. Among the 38 positive lines, 2
lines were obtained from Peak 1 & Peak 2 immunization, 28 lines
were obtained from Peak 1 immunization, 8 lines were obtained
from Peak 2 immunization. CA5-1.23 (gamma-1,lambda,titer 3.27x
10000), CB7-1.1(gamma-1,lambda,titer 5.24x100000), CB7-1.5(gamma-
1,lambda,titer 1.31x100000), CB7-1.19 (gamma-1,lambda,titer 1.31x
100000), EA6-2.2 (gamma-1,kappa,titer 5.24x100000),and CB2-2.18 (
mu,kappa,titer 1.31x100000) were further used to preform Western
blotting and neutralization assay. CA5-1.23, CB7-1.1, CB7-1.5,
CB7-1.19, and EE6-2.2 detected a 60KDa protein among the proteins
in Peak 1. However CB2-2.18 detected four proteins with the M.W.
at 60KDa, 51KDa, 50KDa, 47KDa, respectively. In addition, all six
monoclonal antibodies could partially block the P388D1-mediated
tumoricidal activity against MOPC-315 plasmacytoma. The PIF was
further purified using an immunoaffinity column prepared from
CA5-1.23 coupling Affi-Gel 10. Approximately 1045-fold
purification was achieved and the highly purified PIF showed
strong inhibition against the growth of MOPC-315 plasmacytoma at
a dose as low as 0.625ng.
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