Membrane Proteome Array (MPA)

The MPA was conducted at Integral Molecular, Inc. and is a protein library composed of over 6,000 distinct human membrane protein clones, each overexpressed in live HEK293T cells from expression plasmids. Each clone was individually transfected in separate wells of a 384-well plate followed by a 36-hour incubation.42 Cells expressing each individual MPA protein clone were arrayed in duplicate in a matrix format for high-throughput screening. Before screening on the MPA, the D3-GPC2-IgG1 antibody concentration for screening was determined on cells expressing positive (membrane-tethered Protein A and GPC2) and negative (mock-transfected) binding controls, followed by detection by flow cytometry using a fluorescently-labeled secondary antibody. The D3-GPC2-IgG1 antibody was then added to the MPA at the predetermined concentration, and binding across the protein library was measured on an Intellicyt iQue using a fluorescently-labeled secondary antibody. Each array plate contains both positive (Fc-binding) and negative (empty vector) controls to ensure plate-by-plate reproducibility. D3-GPC2-IgG1 antibody interactions with any targets identified by this MPA screening were validated in a second flow cytometry experiment using serial dilutions of the test antibody, and the target identity was re-verified by sequencing. Figure 3L shows all the validated hits using this approach for the D3-GPC2-IgG1 antibody.