Production and characterization of monoclonal antibodies specific for human mast cell tryptase
- 1 September 1990
- journal article
- Published by Wiley in Clinical and Experimental Allergy
- Vol. 20 (5), 581-589
- https://doi.org/10.1111/j.1365-2222.1990.tb03153.x
Abstract
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.Keywords
This publication has 28 references indexed in Scilit:
- Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetiiCellular Microbiology, 2007
- Cloning and characterization of complementary DNA for human tryptase.JCI Insight, 1989
- Human skin tryptase: purification, partial characterization and comparison with human lung tryptaseBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988
- Tryptase levels in nasal-lavage fluid as an indicator of the immediate allergic responseJournal of Allergy and Clinical Immunology, 1988
- Release of tryptase together with histamine during the immediate cutaneous response to allergenJournal of Allergy and Clinical Immunology, 1987
- Tryptase Levels as an Indicator of Mast-Cell Activation in Systemic Anaphylaxis and MastocytosisNew England Journal of Medicine, 1987
- Localization of tryptase to human cutaneous mast cells and keratinocytes by immunofluorescence and immunoperoxidase cytochemistry with monoclonal antitryptase antibodyJournal of Allergy and Clinical Immunology, 1985
- Quantitative Immunoelectrophoretic Analysis of Rat Allergen ExtractsAllergy, 1983
- The mesothelial keratins: A new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epitheliaCell, 1982
- Histochemical Demonstration of a Species-Specific Trypsin-like Enzyme in Mast CellsNature, 1960