Assessment of the anti-c-kit monoclonal antibody YB5.B8 in affinity magnetic enrichment of human lung mast cells.
The monoclonal antibody, YB5.B8 binds to the second domain of the c-kit proto-oncogene product on human mast cells, a receptor associated with tyrosine kinase activity. This molecule is involved with cell proliferation, maturation and viability as well as cell activation and its natural ligand is stem cell factor (SCF). We have used this antibody coupled to Dynabeads to perform positive affinity enrichment of human lung mast cells. This procedure results in enrichment of mast cells from 2.6 +/- 0.3% to 85.0 +/- 1.6% purity (n = 29) with yields of 41.9 +/- 3.7% (n = 29). As YB5.B8 interacts with the same receptor domain as does SCF, it is important to demonstrate that this procedure does not modify mast cell function. Incubation of mast cells with 1-5000 ng/ml YB5.B8 for 30 min neither induced histamine release nor modulated histamine release induced by anti-IgE. Furthermore, incubation with YB5.B8 did not alter prolonged culture with SCF. Examination of cells enriched using YB5.B8 showed that they had a normal histamine content (3.8 +/- 0.3 pg/cell compared with 3.9 +/- 0.7 pg/cell unpurified, n = 20) and had unchanged behaviour in both histamine secretion and cell survival studies. These studies indicate that YB5.B8 does not influence mast cell function and thus its use in magnetic affinity purification procedures offers a simple and effective method for enriching human mast cell preparations.
Okayama
Y
Y
Immunopharmacology Group, Southampton General Hospital, UK.
Hunt
T C
TC
Kassel
O
O
Ashman
L K
LK
Church
M K
MK
D000911:Antibodies, Monoclonal; D002470:Cell Survival; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000096:biosynthesis; D018189:Immunomagnetic Separation / Q000379:methods; D008168:Lung / Q000166:cytology* / Q000276:immunology; D008407:Mast Cells / Q000166:cytology* / Q000276:immunology; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000276:immunology*; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology*; D016184:Receptors, Colony-Stimulating Factor / Q000276:immunology*
D000911:Antibodies, Monoclonal; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D007073:Immunoglobulin E; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases