Purification and identification of cell surface antigens using lamprey monoclonal antibodies

Abstract

Variable lymphocyte receptor (VLR) B antibodies of the evolutionary distant sea lamprey are structurally distinct from conventional mammalian antibodies. The different protein architecture and large evolutionary distance of jawless vertebrates suggest that VLR antibodies may represent promising tools for biomarker discovery. Here we report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. We demonstrate that despite predicted low affinity of individual VLR antigen binding units to the antigen, the high avidity resulting from decameric assembly of secreted VLR antibodies allows for efficient antigen capture and subsequent identification by mass spectometry. We show that VLR antibodies detect their antigens with high specificity and can be used in various standard laboratory application techniques. The lamprey antibodies are novel reagents that can complement conventional monoclonal antibodies in multiple scientific research disciplines.

Identification of human plasma cells with a lamprey monoclonal antibody

“…To facilitate purification and manipulation of this reagent, we engineered HA- and 6xHis-tags in the conserved stalk region of the molecule, as illustrated in Figure 1B and ref. 28, and deleted the C-terminal 42 residues to generate a secreted, monomeric form of the VLRB MM3 Ab (Figure 1C). …”

“…In contrast, staining of the CD38-EGFP transfectants with conventional anti-CD38 Abs showed an expected direct correlation of staining intensity with CD38-EGFP expression levels. In control experiments, we transfected BJAB cells with plasmids encoding CD5-GFP fusion constructs and analyzed them by flow cytometry using conventional anti-CD5 Abs or with the lamprey CD5-specific VLR32 Ab (28). These experiments also revealed the expected direct correlation of cell-surface staining with increasing amounts of CD5-EGFP fusion protein expression (Figure 5A, right panels), indicating that the threshold-based recognition of CD38 by VLRB MM3 was not a binding characteristic inherent to all VLRB Abs.…”

“…Sea lamprey larvae (10–12 cm in length) were immunized with 5 × 10 6 BM aspirate cells from a patient with multiple myeloma in 0.66× PBS (25, 28). Prior to immunization, BM aspirate cells were subjected to depletion of CD45 + cells using magnetic separation.…”

“…Plasmid DNA was isolated from individual bacterial colonies and used for transfection of HEK293T cells. The VLRB-containing culture supernatant was verified by Western blotting for the presence of monoclonal VLR Abs as described previously (28) and subsequently used for staining of BM aspirate cells. Plasmids encoding monoclonal VLRB Abs recognizing BM aspirate cells were selected for further analysis and their composition determined by DNA sequencing.…”

“…Plasmids encoding monoclonal VLRB Abs recognizing BM aspirate cells were selected for further analysis and their composition determined by DNA sequencing. Monomeric VLRB MM3 was generated by deleting sequences encoding the C-terminal 42 aa of the protein (28). …”