Production and characterization of monoclonal antibodies specific for human mast cell tryptase.
Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.
Walls
A F
AF
Immunopharmacology Group, University of Southampton, U.K.
Bennett
A R
AR
McBride
H M
HM
Glennie
M J
MJ
Holgate
S T
ST
Church
M K
MK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000032:analysis / Q000145:classification; D008168:Lung / Q000166:cytology; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010447:Peptide Hydrolases / Q000032:analysis* / Q000276:immunology; D011817:Rabbits
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase