Laborsaving production of site-specific anti-phospho monoclonal antibodies by simultaneous screening for multiple phosphopeptides using multiplex bead assay (P3372)

Abstract

Abstract Monoclonal antibodies (MAbs) to phosphotyrosines at specific sites of cellular proteins are critical tools to analyze the functional status of proteins. However, time consuming and laborious screening steps in current methods limits production. Here, we introduce a bead-based multiplex flow cytometry method for simultaneous evaluation of the generated MAbs for the cross-reactivity to different peptide sequences. In this proof of concept study, we chose four phosphorylation motifs from different receptors expressed on B cells (Ig-alpha, membrane IgG and IgE, and FCRL5). They were synthesized as a series of 11-mer peptides containing a tyrosine or a phosphotyrosine at the center and surrounded by unique amino acid sequences. They were conjugated with different color-coded beads which can be identified by red laser excitation in flow cytometry. Antibody binding to the peptides was measured as the level of blue laser excitable R-Phycoerythrin fluorescence. Mice immunized with phosphopeptide-KLH conjugates were used for making hybridomas. As expected, the simultaneous screening revealed unique specificities of the 10-30 MAbs generated in each fusion experiment. In particular, we obtained anti-phospho MAbs specific to Ig-alpha and membrane IgG as well as pan anti-phospho tyrosine Ab. Our results suggest that this method is suitable for production of anti-phospho MAbs that need to recognize a specific phosphopeptide among other phosphopeptides with structural similarity.