A non-culture-based leptospirosis vaccine that cross-protects against infection caused by heterologous Leptospira spp. should replace the currently available products, which are qualitatively and quantitatively inadequate. With that in mind, two murine hybridomas secreting monoclonal antibodies (MAb) binding only to homogenates of pathogenic Leptospira spp., and not of the saprophytic L. biflexa, serogroup Patoc, serovar Patoc, were produced. The MAbs of both clones neutralized Leptospira-mediated human red blood cell lysis in vitro and rescued hamsters from lethal infection with heterologous Leptospira spp. The orthologous Leptospira spp. protein carrying the MAb epitope(s) was identified by two-dimensional gel electrophoresis (2DE)-based proteomics and database search. The epitopes of the MAbs were located on the major outer membrane protein LipL32 of the pathogenic Leptospira spp. The MAbs in their humanized version are potential leptospirosis immunotherapeutics. They are also suitable as detection reagents in antigen-based assays for the rapid diagnosis of leptospirosis. Recombinant LipL32 is a good candidate for a broad spectrum, non-culture-based leptospirosis vaccine.