Rapid determination of antigenic epitopes in human NGAL using NMR.
The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.
Advanced Technology, Global Pharmaceutical Research and Development, Abbott, Abbott Park, IL 60064, USA.
Edward T
ET
Olejniczak
Qiaoqiao
Q
Ruan
Robert N
RN
Ziemann
Larry G
LG
Birkenmeyer
Sylvia C
SC
Saldana
Sergey Y
SY
Tetin
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000276:immunology
D000209:Acute-Phase Proteins; D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins