Monoclonal antibodies against urease from Canavalia ensiformis.
Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canavalia ensiformis were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. All culture wells exhibited hybrid growth and 25% of these (ie 45 culture wells) contained anti-urease activity. Two positive hybrid cells were cloned twice by the limiting dilution method and three hybridoma clones (B6F, C4F and B18) secreting monoclonal antibodies were selected at random for purification and characterisation purposes. All three cell lines secreted monoclonal antibodies of IgM class which were purified by gel filtration chromatography on Sephacryl S-200 column with a final recovery of 85% and a purification factor of about 18. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 920,000 Da. mAbs were highly specific for jack bean urease as determined by Western blotting. The affinity constants (K) for these mAbs ranged from 10(8) to 10(9) l mol-1. mAb B6F inhibited about 65% of urease activity whereas C4F and B18 stimulated the enzyme activity slightly by 20%. The presence of 2-mercaptoethanol in incubation mixtures protected urease from inactivation by B6F. Urease inactivation by B6F could be reversed by addition of 2-mercaptoethanol which reactivated most of the partially inactive enzyme. Gel filtration chromatography of purified urease exhibited two protein peaks with M(r) values of 290,000 and 90,000 Da which revealed antibody activity. This result suggests that the mAb B6F recognizes the trimeric as well as the monomeric forms of urease.
Karmali
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Departamento de Engenharia Química do Instituto Superior de Engenharia de Lisboa, Portugal.
Domingos
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D005260:Female; D006825:Hybridomas; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010946:Plants, Medicinal; D014407:Tumor Cells, Cultured; D014510:Urease / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D014510:Urease