Characterization of the interaction of alkaline phosphatase with an activity inhibiting monoclonal antibody by progress curve analysis

• Published in: Journal of immunological methods Vol. 182; no. 1; pp. 29 - 39
• Main Authors: Cumme, Gerhard A, Walter, Uwe, Bublitz, Renate, Hoppe, Horst, Rhode, Heidrun, Horn, Anton
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 1995; Elsevier
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - immunology; Animals; Antibodies, Monoclonal - immunology; Antibody-antigen interaction; Antigen-antibody reactions, antigen-antibody complexes, antibody-complement and others. Study of affinity. Antigen presentation; Biological and medical sciences; Cattle; Enzyme immunoinhibition; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glycosyl-phosphatidylinositol-anchored alkaline phosphatase; Kinetics; Models, Immunological; Molecular immunology; Progress curve analysis; Rate constant determination; Glycosyl-phosphatidylinositol-anchored alkaline phosphatase; Alkaline phosphatase; Antibody-antigen interaction; Progress curve analysis; Rate constant determination; PLC; M-GPI-AP; PtdIns; Enzyme immunoinhibition; GPI-AP; AP; Binding capacity; Structure activity relation; Enzyme; Phosphoric monoester hydrolases; Hydrolases; Molecular interaction; Esterases; Molecular complex; Monoclonal antibody; Mathematical model; Immune complex
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00015-3

Using the enzyme activity inhibiting monoclonal antibody IB 10B8 against alkaline phosphatase of calf intestine (AP), the interaction of a macromolecular antigen with the antibody was studied with different reaction conditions and with different conformations of the antigen, i.e. using (i) different pH values, (ii) different temperatures, (iii) different substrate saturation of the enzyme, (iv) different glycosylphosphatidyl-AP (GPI-AP) aggregates, and (v) membrane-bound species. In the case of antibody excess and negligible substrate consumption enzymic product formation proceeds according to [
P] =
a +
b ·
t −
c · exp(−
d ·
t). By direct progress curve fitting and secondary data evaluation using nonlinear regression, omitting numerical derivation and graphic techniques, kinetic constants of the immune reaction have been estimated. The method does not require any artificial labelling nor any separation of bound and free entities.
(i) Upon increasing pH from 9.8 to 11.0, the dissociation constant of the enzyme-antibody complex is increased strongly, mainly due to the decreasing association rate constant. (ii) A temperature increase from 25° C to 37° C produces a marked increase of both the association and dissociation rate constant. (iii) To differentiate between the interaction of the antibody with the free (
E) and substrate-bound (
ES) enzyme, experiments were done at different substrate concentrations. The results were fitted to a model allowing determination of association and dissociation rate constants of the free and substrate-bound enzyme. The inverse variation of association and dissociation rate constants caused by substrate binding produces a marked increase of the dissociation constant of the antibody-enzyme complex. The antibody-bound enzyme shows a nearly three-fold higher
K
m value and a six-fold lower catalytic constant as compared to the free enzyme. (iv) Investigations of the interaction of the antibody with anchorless AP, different hydrophobic aggregates of purified GPI-AP (fractions II–V). (v) Membrane-bound GPI-AP show that the epitopes of all species are fully accessible to the antibody and not cryptic. Surprisingly the insertion of the GPI-moiety into the membrane and the aggregation of the different GPI-AP fractions II–V seem to improve antibody binding. Such improvement of binding was not found in control experiments with Fab, indicating only for the bivalent antibody a stronger interaction with the multivalent antigen than with the monovalent antigen.