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.