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 i...

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Veröffentlicht in:	Analytical biochemistry 1997-05, Vol.247 (2), p.382-388
Hauptverfasser:	van den Elsen, J M, van Pomeren, E, Poolman, J T, Wilting, J, Herron, J N, Crommelin, D J
Format:	Artikel
Sprache:	eng
Schlagworte:	Amino Acid Sequence Animals Antibodies, Bacterial Antibodies, Monoclonal Antigen-Antibody Reactions Antigens, Bacterial Binding Sites Binding, Competitive Epitopes Fluorescence Polarization - methods Immunoassay - methods Immunoglobulin Fab Fragments In Vitro Techniques Kinetics Mice Molecular Sequence Data Neisseria meningitidis - immunology Peptides, Cyclic - chemistry Peptides, Cyclic - immunology Porins - immunology
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Beschreibung
Zusammenfassung:	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.
ISSN:	0003-2697
DOI:	10.1006/abio.1997.2064