Characterization of ultra-high affinity monoclonal antibodies with a dimeric, symmetrical antigen: inhibition of the receptor recognition site of nerve growth factor.
We generated a family of ultra-high affinity monoclonal antibodies (MAb) which inhibit competitively the binding of nerve growth factor (NGF) to its receptor. Preliminary experiments indicated that the dissociation constants (Kd) of some of the MAb:NGF complexes were substantially less than 0.1 nM. Conventional methods, such as ELISA and radioimmunoassays (RIA), were not sufficiently sensitive to measure the Kds of these MAb. Therefore, experimental conditions were developed to determine binding constants for these very high affinity MAb. The experiments establish that the Kds for our anti-NGF MAb range from 2.6 nM to 39 fM. Additionally, the inhibition of NGF binding to NGF-receptor by MAb is fully consistent with a purely competitive model but is not consistent with a model allowing the formation of a ternary complex of NGF, MAb, and NGF-receptor. One MAb, M4, immunoprecipitates NGF indicating interaction between each protomer of the NGF dimer and individual MAb molecules. We also evaluated the effects of mild denaturing conditions on the binding and biological activity of NGF and on recognition by the MAbs. Guanidine HCl or heat treatment of NGF resulted in only small, but significant, changes in binding or biological activity, in parallel with changes in recognition by the MAbs. However, binding, biological activity, and recognition by six of seven MAbs were completely eliminated by beta-mercaptoethanol reduction. Thus, our results are consistent with the MAbs interacting with the receptor recognition site on the surface of the NGF molecule. The high affinity MAbs will serve as sensitive probes of structural elements of NGF responsible for binding and biological activity.
Vroegop
S M
SM
Cell Biology, Upjohn Company, Kalamazoo, MI 49001.
Crumm
E
E
Buxser
S E
SE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature / Q000009:adverse effects; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000032:analysis; D066298:In Vitro Techniques; D016018:Least-Squares Analysis; D008623:Mercaptoethanol; D051379:Mice; D009414:Nerve Growth Factors / Q000276:immunology*; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000276:immunology*; D017475:Receptors, Nerve Growth Factor
D000911:Antibodies, Monoclonal; D006146:Guanidines; D007074:Immunoglobulin G; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D008623:Mercaptoethanol; D019791:Guanidine