Non-specific cross-reacting antigen: characterization of specific and cross-reacting epitopes.
A cDNA for NCA-50 was cloned into the inducible expression vector pTRB1, using the polylinker site at the C-terminus of the lac Z' gene. An NCA-specific MAb (N1), NCA and CEA cross-reactive MAbs (T84.1, 192) and polyclonal antisera (anti-NCA and anti-CEA, as well as anti-PS beta G) detected the fusion protein, with a mol. wt of 155,000, which constituted about 5% of the total bacterial protein. Deletion and mutation analysis showed that all MAbs which stained positive in western blots mapped to a small region within the last third of the N-terminal domain. Superimposition of the deduced amino acid sequence of NCA-50 on the known structure of immunoglobulins reveals that the antigenic region is located on a surface loop, which corresponds to a fourth hypervariable region on the immunoglobulin heavy chain variable regions. By oligonucleotide directed site-specific mutagenesis amino acids were deduced, which constitute part of an epitope, to which the NCA-50-specific MAb, N1, binds.
Schwarz
K
K
Institut für Immunobiologie, Universität Freiburg, F.R.G.
Brückel
N
N
Schwaibold
H
H
von Kleist
S
S
Grunert
F
F
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000276:immunology; D015815:Cell Adhesion Molecules; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D002272:Carcinoembryonic Antigen; D015815:Cell Adhesion Molecules; D000939:Epitopes; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins