Molecular dissection of an immunodominant epitope in Kv1.2-exclusive autoimmunity.

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.

MARTin-an open-source platform for microarray analysis.

Background: Microarray technology has brought significant advancements to high-throughput analysis, particularly in the comprehensive study of biomolecular interactions involving proteins, peptides, and antibodies, as well as in the fields of gene expression and genotyping. With the ever-increasing volume and intricacy of microarray data, an accurate, reliable and reproducible analysis is essential. Furthermore, there is a high level of variation in the format of microarrays. This not only holds true between different sample types but is also due to differences in the hardware used during the production of the arrays, as well as the personal preferences of the individual users. Therefore, there is a need for transparent, broadly applicable and user-friendly image quantification techniques to extract meaningful information from these complex datasets, while also addressing the challenges posed by specific microarray and imager formats, which can flaw analysis and interpretation. Results: Here we introduce MicroArray Rastering Tool (MARTin), as a versatile tool developed primarily for the analysis of protein and peptide microarrays. Our software provides state-of-the-art methodologies, offering researchers a comprehensive tool for microarray image quantification. MARTin is independent of the microarray platform used and supports various configurations including high-density formats and printed arrays with significant x and y offsets. This is made possible by granting the user the ability to freely customize parts of the application to their specific microarray format. Thanks to built-in features like adaptive filtering and autofit, measurements can be done very efficiently and are highly reproducible. Furthermore, our tool integrates metadata management and integrity check features, providing a straightforward quality control method, along with a ready-to-use interface for in-depth data analysis. This not only promotes good scientific practice in the field of microarray analysis but also enhances the ability to explore and examine the generated data. Conclusion: MARTin has been developed to empower its users with a reliable, efficient, and intuitive tool for peptidomic and proteomic array analysis, thereby facilitating data-driven discovery across disciplines. Our software is an open-source project freely available via the GNU Affero General Public License licence on GitHub.