Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells.
Two murine monoclonal antibodies were prepared against tryptase, the major neutral protease and protein component of human mast cells. The antibodies were termed G5 (IgG2B-kappa) and H4 (IgG1-kappa). They were specific for tryptase by an enzyme-linked immunosorbent assay and an immunotransblot technique. The latter procedure showed that H4 and G5 each bind to the 35,000 and 37,000 m.w. subunits of tryptase, indicating immunologic cross-reactivity between the subunits. The monoclonal antibodies reacted only with tryptase subunits in an extract of dispersed lung cells. By immunofluorescence microscopy, tryptase was further identified to be present only in cytoplasmic granules of Alcian Blue-stained mast cells in dispersed pulmonary cell preparations. No evidence for a mast cell subtype lacking tryptase was detected. In addition, a procedure for the purification of tryptase to homogeneity from dispersed pulmonary cells containing less than 10% mast cells was developed; this procedure involved high salt extraction, ammonium sulfate precipitation, and sequential chromatography with decyl-agarose, DEAE-agarose, and heparin-agarose. The procedure resulted in a higher yield even with less pure starting material than reported previously. Tryptase is a selective marker for mast cells in dispersed pulmonary cells, and can be detected with specific anti-tryptase antibodies.
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D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D006801:Humans; D046911:Macromolecular Substances; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase