Specificity and functional effects of antibodies to human stem cell factor.
Three monoclonal antibodies (Mabs), 7H6, 4B10 and Genzyme Mab, and a commercially-available polyclonal antiserum (Genzyme) to human Stem Cell Factor (SCF) were compared for their ability to detect native and recombinant SCF in a variety of assays, and for blocking of SCF function. All antibodies were found to bind to the membrane bound isoform as well as soluble SCF and to bind to both glycosylated (yeast MGF) and unglycosylated (E. coli SCF) recombinant factor. Mabs 7H6 and 4B10, as well as the polyclonal antiserum could immunoprecipitate membrane-associated SCF and all the antibodies could detect recombinant soluble SCF on western blots, although the binding of all except 7H6 was partially sensitive to reduction. Titration of the antibodies on CHO cells expressing membrane-associated human SCF showed similar dose-dependence for all Mabs with 70% of maximum binding seen at 3, 5 and 8 micrograms/ml for 7H6, 4B10 and Genzyme Mab respectively, however the maximum binding seen with 7H6 was approximately 2-fold greater than with 4B10 and 7-fold greater than Genzyme Mab. Competitive binding experiments of the Mabs on cells expressing membrane SCF gave non-reciprocal blocking in all cases with 7H6 completely blocking 4B10 and Genzyme Mab binding. All antibodies except the Genzyme Mab effectively blocked SCF binding to c-Kit-expressing cells, and were strongly inhibitory in an assay of in vitro haemopoiesis which is believed to depend on adhesive interactions, as well as the "classical' cytokine-receptor interaction, mediated by SCF binding to c-Kit.
Zannettino
A C
AC
Leukaemia Research Unit, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia.
Aylett
G W
GW
Leavesley
D I
DI
Pietsch
T
T
Chang
D G
DG
Simmons
P J
PJ
Ashman
L K
LK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001667:Binding, Competitive; D015153:Blotting, Western; D001854:Bone Marrow Cells; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006410:Hematopoiesis; D006801:Humans; D011233:Precipitin Tests; D011485:Protein Binding; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D019089:Stem Cell Factor / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit