Characterization of Monoclonal Antibodies Against Carcinoembryonic Antigen (CEA) and Expression in E. coli

Abstract

Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG₁, two clones are IgM and one clone is IgG_2b; all have kappa light chain. The affinities are in the range of 1.1 × 10^-7 ~ 2.4 × 10^-9 M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of V_H and V_L of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 × 3.4-fold lower affinities (2.8 × 10^-8 ~ 3.6 × 10^-9 M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited ~ 10^-6 M of affinity.