title: Characterization of monoclonal antibodies against rat glandular kallikrein.
abstract: Monoclonal antibodies can be produced in large amounts, are homogenous and can be highly purified. A specific monoclonal antibody against glandular kallikrein could be very useful in studies of the kallikrein-kinin system, both in vivo and in vitro. Two monoclonal antibodies against rat glandular kallikrein (rgKK) were produced by immunized mouse spleen and lymph node fusion with myeloma Ag8.653. Both antibodies, named 2E9.8 and 2E9.9, bound active 125I-kallikrein and phenylmethylsulfonyl fluoride (PMSF)-inactivated 125I-kallikrein. A radioimmunoassay (RIA) was developed with each of the antibodies using rabbit anti-mouse gamma globulin to separate bound from free 125I-rgKK. The standard curve (range 10-1000 ng/tube) was curved even when subjected to logit-log transformation. Using 3% polyethylene glycol (PEG) to assist separation of bound from free, the standard curve became straight for 2E9.8 and the RIA was more sensitive, with a binding range of 0.35-2.4 ng/tube. Both antibodies were specific for rgKK since they had negligible cross-reaction with purified proteases from the submandibular gland of the rat (tonin, esterases B and E). They did not cross-react with mouse nerve growth factor, epidermal growth factor, nor with pig pancreatic kallikrein. Antibody 2E9.9 did appear to bind some human kallikrein when tested with high concentrations of this enzyme, while 2E9.8 did not. When preincubated with purified rgKK, both antibodies prevented the enzyme from releasing kinins from semi-purified dog kininogen and from cleaving [3H]-L-arginine methyl ester (3H-TAME). These results suggested that both antibodies bind an epitope near to, and maybe including, the active site of the enzyme. Monoclonal antibody 2E9.8 appears to be specific for rgKK, can be used in a sensitive RIA, and is capable of inhibiting the enzymatic activity of kallikrein. It should prove to be useful in vivo for examining the role of kallikrein in physiological processes.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D004950:Esterases / Q000037:antagonists & inhibitors; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000276:immunology*; D011863:Radioimmunoassay; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D004950:Esterases; D007610:Kallikreins