On the interaction between a bactericidal antibody and a PorA epitope of Neisseria meningitidis in outer membrane vesicles: a competitive fluorescence polarization immunoassay.
This paper describes a method for determining the affinity constant (Ka) of the binding between an antibody Fab fragment and a membrane-embedded protein epitope under equilibrium conditions. Monoclonal antibody MN12H2, directed against outer membrane protein PorA of Neisseria meningitidis, is used in a competitive fluorescence polarization assay with a cyclic peptide-fluorescein conjugate as a tracer antigen. Displacement experiments with PorA-containing and PorA-deficient meningococcal outer membrane vesicles revealed highly specific binding of MN12H2 Fab to the membrane-embedded PorA P1.16 epitope with Ka of 1.5 x 10(8) M-1.
van den Elsen
J M
JM
Department of Pharmaceutics, Faculty of Pharmacy, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, The Netherlands.
van Pomeren
E
E
Poolman
J T
JT
Wilting
J
J
Herron
J N
JN
Crommelin
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial; D001665:Binding Sites; D001667:Binding, Competitive; D000939:Epitopes; D005454:Fluorescence Polarization / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000276:immunology*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000276:immunology; D018272:Porins / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D010456:Peptides, Cyclic; D018272:Porins; C057699:porin protein, Neisseria