Production and Characterization of Monoclonal Antibodies for Immunoassay of the Lung Cancer Marker proGRP.
Progastrin-releasing peptide (proGRP) is a precursor of gastrin-releasing peptide, a hormone which is secreted from neuroendocrine cells. It has been shown to be a useful serum marker for small cell lung cancer. We raised monoclonal antibodies (MAbs) against proGRP with the primary aim of establishing a sensitive immunoassay. Immunization was performed with recombinant proGRP (amino acids 31-98) conjugated to thyroglobulin or with a DNP-modified peptide. Seven of the MAbs recognizing both recombinant and cell line-derived peptide were characterized and epitope-mapped. Based on cross-inhibition studies the antibodies could be categorized into three main groups. The molecular epitope assignment was studied by using phages displaying proGRP peptides, random peptide libraries displayed on phage and by pepscan analysis utilizing 10-mer biotinylated peptides. Two of the MAbs (E146, E172) bound to a defined region on the N-terminal part of proGRP(31-98), three recognized conformational-dependent epitopes in the middle of the peptide (E179, E180, E181) and two bound to the C-terminal part (E149, E168). Consensus sequences were obtained for MAbs E146, E149 and E168. The binding kinetics of the MAbs was determined by surface plasmon resonance, and a time-resolved immunofluorometric assay was established.
Nordlund
Marianne S
MS
Department of Medical Biochemistry, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway. marisno@ulrik.uio.no
Fermer
Christian
C
Nilsson
Olle
O
Warren
David J
DJ
Paus
Elisabeth
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000276:immunology*; D018288:Carcinoma, Small Cell / Q000276:immunology*; D004140:Dinitrophenols / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007118:Immunoassay; D008175:Lung Neoplasms / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D019151:Peptide Library; D010455:Peptides / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D004140:Dinitrophenols; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; C087079:pro-gastrin-releasing peptide (31-98); C024118:DNP-peptide