Please use this identifier to cite or link to this item: https://ahro.austin.org.au/austinjspui/handle/1/13342
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dc.contributor.authorTrapani, Joseph Aen
dc.contributor.authorBrowne, K Aen
dc.contributor.authorDawson, M Jen
dc.contributor.authorSmyth, Mark Jen
dc.date.accessioned2015-05-16T03:10:33Z
dc.date.available2015-05-16T03:10:33Z
dc.date.issued1993-09-15en
dc.identifier.citationBiochemical and Biophysical Research Communications; 195(2): 910-20en
dc.identifier.govdoc8373425en
dc.identifier.otherPUBMEDen
dc.identifier.urihttp://ahro.austin.org.au/austinjspui/handle/1/13342en
dc.description.abstractEnzymatically 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.en
dc.language.isoenen
dc.subject.otherAmino Acid Sequenceen
dc.subject.otherAnimalsen
dc.subject.otherAntibodies, Monoclonalen
dc.subject.otherAntigen-Antibody Complex.isolation & purificationen
dc.subject.otherBlotting, Westernen
dc.subject.otherCloning, Molecularen
dc.subject.otherElectrophoresis, Polyacrylamide Gelen
dc.subject.otherEnzyme-Linked Immunosorbent Assayen
dc.subject.otherGranzymesen
dc.subject.otherHumansen
dc.subject.otherKiller Cells, Natural.enzymologyen
dc.subject.otherLeukemiaen
dc.subject.otherLeukemia, Experimentalen
dc.subject.otherMiceen
dc.subject.otherMolecular Sequence Dataen
dc.subject.otherMolecular Weighten
dc.subject.otherPeptides.chemical synthesis.immunologyen
dc.subject.otherRatsen
dc.subject.otherRecombinant Proteins.biosynthesis.immunology.isolation & purificationen
dc.subject.otherSerine Endopeptidases.biosynthesis.immunology.isolation & purificationen
dc.subject.otherTransfectionen
dc.subject.otherTumor Cells, Cultureden
dc.titleImmunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody.en
dc.typeJournal Articleen
dc.identifier.journaltitleBiochemical and biophysical research communicationsen
dc.identifier.affiliationCellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Australiaen
dc.identifier.doi10.1006/bbrc.1993.2131en
dc.description.pages910-20en
dc.relation.urlhttps://pubmed.ncbi.nlm.nih.gov/8373425en
dc.type.austinJournal Articleen
item.grantfulltextnone-
item.cerifentitytypePublications-
item.fulltextNo Fulltext-
item.openairetypeJournal Article-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en-
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