Characterization of Monoclonal Antibodies Recognizing 130 kDa Surface Proteins on Human Embryonic Stem Cells and Cancer Cell Lines

Abstract

To study cell surface proteins expressed on human embryonic stem cells (hESCs), we generated a panel of monoclonal antibodies (MAbs) against undifferentiated hESCs by a decoy immunization strategy in a previous study. Two of the MAbs, 63-B6 and 246-D7, bound to human pluripotent stem cells but not to human primary cells such as human peripheral blood mononuclear cells and human lung fibroblasts. They did not bind to either mouse embryonic stem cells or mouse embryonic fibroblasts. The two MAbs had similar binding profiles for many various cancer cells, with few exceptions. Expression of antigens recognized by the two MAbs was rapidly decreased during embryoid body formation of hESCs and gradually increased after initial decrease. The MAbs recognized approximately 130 kDa proteins on the surface of hESCs. Cloning and sequence analysis of antibody genes showed that although the MAbs had exactly the same light chain sequences, they had different heavy chain sequences. Taken together, the results suggest that the two MAbs may recognize two different epitopes of the same or different 130 kDa surface proteins involved in regulating the early differentiation of human pluripotent stem cells and cancer cells.

Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31

“…Heavy and light chain cDNAs were generated from RNAs by using One-Step RT-PCR PreMix Kit (iNtRON Biotechnology, Seoul, Korea), according to the manufacturer’s protocol. The coding regions were amplified by polymerase chain reaction (PCR) using specific primers, described as a previous study [17]. The amplified cDNAs were cloned into pBluescript cloning vector using the EcoRI/SalI or HindIII/SalI restriction enzyme sites.…”

B-Cell Receptor-Associated Protein 31 Regulates Human Embryonic Stem Cell Adhesion, Stemness, and Survival via Control of Epithelial Cell Adhesion Molecule

“…MAb 297‐D4 was purified from the culture supernatant of hybridoma by Protein G‐Sepharose column chromatography as described previously and biotinylated as described previously .…”

Antibody approaches to prepare clinically transplantable cells from human embryonic stem cells: Identification of human embryonic stem cell surface markers by monoclonal antibodies

“…This approach is a promising tool to identify and characterize new surface antigens expressed by hESCs. In some cases, the identities of antigens on hESC remain unknown [40,41,53,54,68,69] although the presence of the antigens is proven by mAbs. Thus, the identification of antigens recognized by the mAbs is the primary rate-limiting step in this antibody-based approach.…”