General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice
Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2+CD80+ memory B cells, with significantly elevated CD69 and CD86 observed in RBP+ marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.
Combinatorial Synthesis of Peptide Arrays onto a Microchip
Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.
Multifunctional CMOS Microchip Coatings for Protein and Peptide Arrays
Stadler, Volker; Beyer, Mario; König, Kai; Nesterov, Alexander; Torralba, Gloria; Lindenstruth, Volker; Hausmann, Michael; Bischoff, F. Ralf; Breitling, Frank
Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules. The coated microchips were compatible with the harshest conditions emerging in microarray generating methods, thoroughly retaining structural integrity and microelectronic functionality. Nonspecific adsorption of proteins on the chip’s surface was completely obviated even with complex serum protein mixtures. We could demonstrate the background-free antibody staining of immobilized probe molecules without using any blocking agents, encouraging further integration of CMOS technology in proteome research.
