Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody.
Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO(3)H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds.
Lassen
Klaus S
KS
Department of Autoimmunology and Immunological Development, Statens Serum Institut, Copenhagen S, Denmark.
Bradbury
Andrew R M
AR
Rehfeld
Jens F
JF
Heegaard
Niels H H
NH
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D000918:Antibody Specificity; D003181:Complement C4 / Q000737:chemistry / Q000276:immunology; D019075:Electrophoresis, Capillary; D005755:Gastrins / Q000737:chemistry / Q000276:immunology; D006629:Hirudins / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D012844:Sincalide / Q000737:chemistry / Q000276:immunology; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D003181:Complement C4; D005755:Gastrins; D006629:Hirudins; D007074:Immunoglobulin G; D010446:Peptide Fragments; C005662:tyrosine O-sulfate; D014443:Tyrosine; C013550:gastrin 17; D012844:Sincalide