MONOCLONAL-ANTIBODIES AGAINST UREASE FROM CANAVALIA-ENSIFORMIS

Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canava lia 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 monoclo nal antibodies were selected at random for purification and characteri sation purposes. All three cell lines secreted monoclonal antibodies o f IgM class which were purified by gel filtration chromatography on Se phacryl S-200 column with a final recovery of 85% and a purification f actor of about 18. The purified preparations were apparently homogeneo us 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 a ffinity constants (K) for these mAbs ranged fro 10(8) to 10(9) l mol-1 . mAb B6F inhibited about 65% of urease activity whereas C4F and B18 s timulated the enzyme activity slightly by 20%. The presence of 2-merca ptoethanol in incubation mixtures protected urease from inactivation b y B6F. Urease inactivation by B6F could be reversed by addition of 2-m ercaptoethanol which reactivated most of the partially inactive enzyme . Gel filtration chromatography of purified urease exhibited two prote in peaks with M(r) values of 290 000 and 90 000 Da which revealed anti body activity. This result suggests that the mAb B6F recognizes the tr imeric as well as the monomeric forms of urease.