Lyme-Borreliosis Disease: IgM Epitope Mapping and Evaluation of a Serological Assay Based on Immunodominant Bi-Specific Peptides

Lyme borreliosis (LB) is a tick-borne infection of global relevance that remains underrecognized, hindering effective surveillance and diagnosis. This lack of awareness and the limited specificity and low antibody titters of current serological assays underscore the need for improved diagnostic tools. Here, we investigated the molecular fine specificity of IgM antibody responses to five proteins of <i>Borrelia burgdorferi</i>. <b>Materials and Methods:</b> We employed peptide arrays on cellulose support (SPOT synthesis) to screen IgM epitopes and assess cross-reactivity through databank searches and Enzyme-Linked Immunosorbent Assay (ELISA). Validation was performed using ELISA and Receiver Operating Characteristic (ROC) curve analysis. <b>Results:</b> We identified ten IgM epitopes, of which four were classified as specific. The ELISA peptide assay demonstrated a sensitivity of ≥87.3%, specificity of ≥56.2%, and accuracy of ≥66.6%. A bi-specific peptide was subsequently synthesized and evaluated by ELISA using a panel of patient sera representing different pathologies. This result showed a sensitivity of 85.0% and a specificity of 100.0%, with significant differences in cross-reactivity between the leptospirosis and syphilis groups. <b>Conclusions:</b> These findings indicate that the identified peptide combinations could facilitate the development of new, highly specific serodiagnostic assays, thereby enhancing public health initiatives and epidemiological studies.