Background : Lyme disease is the most common tick-born infectious disease in Europe. This infection is caused by spirochetes of Borrelia species. Currently, diagnostics are exclusively based on symptom analysis and serological tests. These tests present a lack of sensitivity, specificity and standardization. A more reliable and accurate test is thus needed. In this context, our objective is to develop a direct diagnostic test based on detection of two early bacterial markers of the disease using monoclonal antibodies. We focused on the outer surface proteins C (OspC), a 22kDa lipoprotein that is highly variable between Borrelia species and on the flagellin B (FlaB), a 41kDa major periplasmic flagellum filament. Methods : To detect all OspC variants, we decided to produce monoclonal antibodies (mAbs) directed against a highly conserved OspC motif by immunization of mice with synthetic peptide coupled to a carrier protein. For FlaB, as it presents a high sequence homology between Borrelia burgdorferi strains, we produced antibodies by immunization with a recombinant protein. mAbs were characterized for their affinity using bio-layer interferometry biosensor technology and their sensivitity were evaluated by immunoassay and Western-blot. The capacity of our antibodies to detect bacterial markers was investigated on Borrelia infected mice tissues extract and serum. Mice tissue proteins were extracted, and then enriched by on beads immuno-precipitation and analyzed by Western-blot. Results : 8 mAbs against the conserved OspC peptide and 20 mAbs against the FlaB were produced. During the characterization process, the mAb affinity (Kd) for OspC was determined at 9,31E-10 molar for the antibody of higher affinity, using recombinant OspC. We selected the mAb OspC-11, allowing reaching a limit of detection close to 50pg of OspC protein by WesternBlot. For FlaB detection, the mAb FlaB-15 was selected detecting as low as 10 bacteria by Western-Blot. Both of these early markers were detected in infected mice samples. Conclusions :Taking into account the variability of Borrelia species, these new mAbs could be very useful for the diagnosis of Lyme disease at early stage. The next step is to evaluate our methods of detection in other animal models of infection and in clinical patient samples.