Production and characterization of monoclonal antibodies specific for human mast cell tryptase

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.

Two distinct forms of human mast cell chymase

“…Chymase activity during purification was monitored by the hydrolysis of 0.7 mM N-succinyl-Ala-Ala-ProPhe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-NH-Np) in 1.5 M NaCl, 0.79% dimethylsulphoxide, 0.3 M Tris adjusted to pH 8.0 with HCl [31]. Tryptase activity was determined with either 0.90 mM N-benzoyl-DL-arginine p-nitroanilide (routine analysis of chromatography fractions) or with the 40-fold more sensitive substrate 0.5 mM 5-oxoprolyl-Pro-Arg-p-nitroanilide in 1 M glycerol, 0.1 M Tris/HCl, pH 8.0, 1.0 % dimethylsulphoxide [32,37], and elastase was assayed with 0.7 mM N-succinyl-Ala-AlaAla-p-nitroanilide in the same buffer. All assays were performed in a total reaction volume of 100 µl in microtitre plates using the kinetics programme of a Dynatech MR5000 or a ThermoMAX (Molecular Devices) platereader [31,34].…”

Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson’s Disease

“…and mast cell tryptase were provided by Dr Walls (Aslam et al, 2002;Walls et al, 1990aWalls et al, , 1990b) and others were bought from reputable companies, except the polyclonal antibody against serpin-A13 which was made to order by Antibody Production Services Ltd. using a synthetic peptide specific to serpin-A13 as the immunogen. Specificity of the antibodies (except serpin-A13) was determined by the supplier by western blot and immunohistochemistry.…”

NF-κB and TNF-α: A Positive Autocrine Loop in Human Lung Mast Cells?

“…Nucleated cells were then incubated with the anti-c-kit mAb YB5.B8 (13,14) (donated by Dr. L. K. Ashman, Institute of Medical and Veterinary Science, Adelaide, South Australia), washed, and subjected to positive magnetic affinity using goat anti-mouse IgG-coated Dynabeads (15). This procedure yielded mast cells, identified by the mAb AA1 against tryptase (16), in purities of Ͼ95%. Following purification, to allow the cells to recover the rigors of purification and to sensitize them, mast cells were incubated for 16 h with human myeloma IgE (3 g/ml; Calbiochem-Novabiochem, San Diego, CA).…”