Production of a monoclonal antibody that recognizes bovine stem cell factor (SCF) and its use in the detection and quantitation of native soluble bovine SCF in fetal bovine serum.
Stem cell factor (SCF) is a pluritropic hematopoietic cytokine that acts predominantly on the proliferation and differentiation of hematopoietic progenitor cells. SCF has long been thought to be present in fetal bovine serum (FBS) as an endogenous factor that stimulates the growth of hematopoietic progenitor cells in FBS-supplemented cultures. To detect and quantitate bovine SCF in FBS, we produced a monoclonal antibody (mAb) by immunizing mice with recombinant soluble bovine SCF protein, which was expressed in insect cells by using a baculovirus system. Using the mAb, we purified native soluble bovine SCF from FBS by immunoaffinity chromatography. Western blot analysis revealed that the purified SCF protein had a molecular weight of 33 kDa. In addition, an enzyme-linked immunosorbent assay (ELISA) incorporating the mAb revealed that the levels of native soluble SCF in commercially available FBS were likely to be <100 pg/ml. These results suggest that the concentration of native soluble bovine SCF present in FBS may be insufficient to promote additive biologic effects on the growth of hematopoietic progenitor cells in FBS-supplemented cultures.
Hikono
Hirokazu
H
National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan. hikono@affrc.go.jp
Zhou
Jian-Hua
JH
Ohta
Masato
M
Inumaru
Shigeki
S
Momotani
Eiichi
E
Sakurai
Michiharu
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005312:Fetal Blood / Q000737:chemistry*; D007313:Insecta / Q000166:cytology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D012995:Solubility; D019089:Stem Cell Factor / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D019089:Stem Cell Factor