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Title: Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody.
Austin Authors: Trapani, Joseph A;Browne, K A;Dawson, M J;Smyth, Mark J
Affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Australia
Issue Date: 15-Sep-1993
Publication information: Biochemical and Biophysical Research Communications; 195(2): 910-20
Abstract: Enzymatically active granule-associated serine protease ("granzyme") B has been purified from human NK cell lysates, using novel granzyme B-specific monoclonal antibodies. Two antibodies, designated 2C5 and 1D10, were produced following immunization of BALB/c mice with a nineteen amino acid peptide synthesized according to the sequence deduced from a granzyme B cDNA clone. Of several peptide-reactive culture supernatants that resulted from cell fusion of splenocytes with NS-1 myeloma cells, clones 2C5 (IgG2a) and 1D10 (IgG1) produced antibodies which detected a approximately 32kDa molecule in human NK cell lysates by Western blotting. This reactive species was detectable in lysates of IL-2-stimulated peripheral blood mononuclear cells, the human NK leukemia cell line YT, the rat NK leukemia cell line RNK-16, but not in the mouse cytotoxic T cell line CTLL-R8 or a variety of non-cytolytic hemopoietic tumor cell lines. The specificity of reactivity with granzyme B was demonstrated by the reaction of the monoclonal antibody with active granzyme in the lysate of COS-7 cells transfected with human granzyme B cDNA, but not with granzyme H expressed in an identical fashion. Western blotting on Percoll-fractionated IL-2 activated human peripheral blood lymphocyte lysates and YT demonstrated reactivity of the monoclonal antibody with a approximately 32kDa species only in those fractions with granzyme A (BLT esterase) and B (Asp-ase) activities. Moreover, 2C5/1D10 antibodies coupled to Protein A-sepharose beads immunoprecipitated enzymatically active granzyme B from YT cell lysates. Scale up of this procedure should yield a means of purifying the large quantities of natural or recombinant granzyme B required to study the function of this granzyme in cellular cytotoxicity.
Gov't Doc #: 8373425
DOI: 10.1006/bbrc.1993.2131
Journal: Biochemical and biophysical research communications
Type: Journal Article
Subjects: Amino Acid Sequence
Antibodies, Monoclonal
Antigen-Antibody Complex.isolation & purification
Blotting, Western
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Killer Cells, Natural.enzymology
Leukemia, Experimental
Molecular Sequence Data
Molecular Weight
Peptides.chemical synthesis.immunology
Recombinant Proteins.biosynthesis.immunology.isolation & purification
Serine Endopeptidases.biosynthesis.immunology.isolation & purification
Tumor Cells, Cultured
Appears in Collections:Journal articles

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