title: Determination of rate constants of monoclonal antibodies to enzymes.
abstract: A novel graphical method for determining rate constants of the immune reaction of enzyme-inhibiting or -activating antibodies has been evaluated. The experimental determination of kinetic constants does not require purification of antibody and enzyme nor any separation step of bound and free entities. The resultant enzyme activity is used as a measurement of the extent of enzyme-antibody complex formation. The plot ln magnitude of v - v infinity versus t (v = enzyme activity at time t, v infinity = enzyme activity at infinite time) yields straight lines in the case of antibody excess. Slope and vertical intercept of that primary plot can be used for secondary plots to obtain association (k1) and dissociation (k-1) rate constant, the dissociation constant KD of the complex and the residual enzyme activity of the complex (g/f). The suitability of the graphical method has been established experimentally using the mab IB 10B8 (2) which inhibits alkaline phosphatase activity. With the homogeneous assay in the presence of substrate as well as a microassay in the absence of substrate, k1 and g/f were found to be 4.68 x 10(7) M-1 min-1; 3.74 x 10(7) M-1 min-1 and 0.035; 0.107 respectively. For k-1 and KD, only crude estimates could be derived. The method has been tested for model discrimination using computer simulated data.
authors.affiliation: Institute of Biochemistry, Friedrich Schiller University, Jena, G.D.R.
authors.forename: G A
authors.initials: GA
authors.lastname: Cumme
authors.forename: R
authors.initials: R
authors.lastname: Bublitz
authors.forename: H
authors.initials: H
authors.lastname: Ehle
authors.forename: A
authors.initials: A
authors.lastname: Horn
mesh_terms: D000469:Alkaline Phosphatase / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D002417:Cattle; D004789:Enzyme Activation; D007118:Immunoassay; D007158:Immunologic Techniques; D007700:Kinetics; D008433:Mathematics
chemical_list: D000911:Antibodies, Monoclonal; D000469:Alkaline Phosphatase