designed and generated the 2W:IAb MAb. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptideCMHCII complexes. The general approach for generation of monoclonal antibodies (MAb) reactive to a defined protein antigen has been well documented since the original report in 1975 by Drs. Kohler and Milstein1. The utility and broad use of MAbs in biological systems earned Kohler and Milstein the Nobel Prize for medicine in 1984 (ref. 2). In this report we describe a novel methodology to specifically and reliably generate MAbs that target peptide in the context of MHCII, which has only occurred a few times since 1975 (refs 3, 4, 5, 6, 7, 8, 9). To generate a MAb using the traditional approach, mice are immunized, the responding B cells are isolated, fused to myeloma cells with hypoxanthineCaminopterinCthymidine (HAT)-based selection, screened and sub-cloned to isolate monoclonal hybridomas2. Screening requires the examination of hundreds or even thousands of clones for one MAb, creating a major bottleneck. This approach typically yields 1C5% hybridomas specific for a protein target antigen causing a prominent hurdle, both in time and resources4. However, this method is not specifically designed to generate peptide:MHCII (p:MHCII) reactive MAbs, and B-cell tolerance against self MHC adds to the difficulty. To overcome this, we developed a novel methodology to generate MAb against a specific p:MHCII complex. B-cell clones specific for the antigen of interest are enriched immediately before myeloma fusion, thus significantly reducing the screening required. The basis for this methodology centers on having a stable p:MHCII monomeric protein linked to biotin as the B-cell antigenic target. This approach has several advantages. First, immunization with p:MHCII complexes induces a B-cell response specific for that peptide in the context of MHCII. Second, use of antigen-specific tetramers allows us to pre-screen immunized mice to confirm the expansion of p:MHCII-specific B cells. Third, it offers the ability to enrich for antigen-specific B cells10 while discarding B-cell clones responding to unrelated antigens. Specifically, the utility of a site-directed protein biotinylation allows for the enrichment of B cells reactive to the target protein/peptide by generating a tetrameric antigen, thus increasing the avidity of B cells for antigen and enabling the capture and enrichment of antigen-specific B cells10,11. This results in a significant time and cost saving as fewer colonies are required for screening, and a higher percentage of selected hybridomas produce MAb against p:MHCII. Finally, this enrichment approach could be used for any MAb protein target including peptides and haptens, not just p:MHCII12,13,14. Results Generation of p:MHCII MAb The workflow for this methodology and the necessary steps for p:MHCII MAb generation are illustrated in Fig. 1. Generation and validation of p:MHCII MAb BAM 7 can be completed in 8 weeks. We were interested to develop a reagent to block T-cell receptor (TCR) recognition of a diabetes-relevant peptide14,15,16. We initially developed antibodies against p63 peptide in the context of IAg7 MHCII molecule, given that p63-activated BDC2.5 CD4+ T cells mediate accelerated autoimmune diabetes when transferred into wild-type non-obese diabetic (NOD) hosts17,18,19. We isolated splenocytes from five p:MHCII (p63:IAg7) immunized BALB/c mice and magnetically enriched for antigen-specific B cells using PE-conjugated p63:IAg7 tetramers followed by anti-PE magnetic beads10. To validate successful priming and expansion, we analyzed the phenotype of p:MHCII-specific B cells in naive mice compared Rabbit Polyclonal to SPTBN5 to day 7 post immunization (Fig. 2a). Antigen-specific B cells were identified from immunized mice by p:MHCII-PE tetramer excluding those that bound to streptavidin (SA)-phycoerythrin (PE) or SA-allophycocyanin (APC) using SA-PE-AF647 or SA-APC-DyLight 755, compared to a decoy p:MHCII-APC reagent (Fig. 2a). Three distinct subsets of antigen-specific B cells (p:MHCII specific, MHCII specific and decoy p:MHCII specific)10,20 were evaluated for GL7 and intracellular Ig expression associated with mature germinal center B cells. Phenotypic analysis demonstrates the p:MHCII-PE+ population is enriched for mature germinal center B cells (GL7+ and intracellular Ig?) demonstrating successful priming and T-cell help (Fig. 2a). We verified the enrichment approach at day 3 post antigen boost, before hybridoma fusion. Magnetic enrichment resulted in an increase to 11.1% of the B cells staining positive BAM 7 for p63:IAg7-PE tetramer, and phenotypic markers demonstrating the presence of germinal center B cells within this population (Fig. 2b). The enriched fraction contained 2.1 107 cells, which was 23-fold reduced compared with the starting population. These cells were subsequently fused BAM 7 with SP2/0 myeloma cells and plated onto ten, 100?mm plates containing semi-solid media under HAT selection..