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Previously, we\n                        identified the pathogen-specific paralogous PF07598 gene family encoding\n                        virulence-modifying (VM) exotoxins, which play a critical role in\n                        leptospirosis pathogenesis. In this study, we developed a monoclonal\n                        antibody (mAb)-based capture immunoassay that detects VM proteins in the\n                        blood of experimental hamster models, validating the hypothesis that VM\n                        proteins function as secretory exotoxins that mediate disease pathogenesis.\n                        Monoclonal antibodies were generated against a natural variant, LA0591, a VM\n                        protein containing a conserved C-terminal DNase toxin domain but lacking\n                        N-terminal ricin B-like lectin domains. Epitope mapping identified specific\n                        linear epitopes targeted by mAbs 5F8, 5G10, and 6A5, with distinct binding\n                        regions confirmed through binning and peptide mapping. These mAbs\n                        demonstrated high-affinity binding to homologous antigens, with\n                        sub-picomolar dissociation constants (K\n                      <jats:sub>d<\/jats:sub>\n                      = 1.41E-09 for 5F8 and\n                            K\n                      <jats:sub>d<\/jats:sub>\n                      &lt;1.0E-12 for 5G10 and 6A5) and cross-reacted with\n                        full-length recombinant VM proteins expressed in\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      .\n                        Immunoblotting revealed increased expression of VM proteins by\n                      <jats:italic toggle=\"yes\">L.\n                            interrogans<\/jats:italic>\n                      serovar Copenhageni strain L1-130 under\n                      <jats:italic toggle=\"yes\">in\n                            vivo<\/jats:italic>\n                      -like conditions. Using mAbs 6A5 and 5F8, a capture ELISA\n                        detected circulating VM proteins in the serum and urine from infected\n                        hamsters, confirming the secretion of these proteins during infection. This\n                        study provides the first evidence of secreted leptospiral exotoxins in the\n                        bloodstream of infected animals, advancing the understanding of\n                        leptospirosis pathogenesis and establishing a basis for developing novel\n                        diagnostic approaches.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>This research addresses the global health issue of leptospirosis, a neglected\n                        tropical disease that still lacks early and reliable diagnostic methods\n                        despite being known for over a century. The study has developed a novel test\n                        using specially designed antibodies to detect specific proteins related to\n                        the disease in the blood of infected hamsters. These proteins are linked to\n                        the pathogen's ability to cause illness. The successful detection of these\n                        proteins in the bloodstream is a significant advancement, as it not only\n                        improves our understanding of the disease's progression but also lays the\n                        groundwork for developing new diagnostic tools. This could lead to earlier\n                        and more accurate diagnoses of leptospirosis, potentially saving lives and\n                        reducing the impact of the disease globally.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00018-25","type":"journal-article","created":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T13:00:53Z","timestamp":1759150853000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Development of leptospiral virulence-modifying protein detection                    assay: implications for pathogenesis and diagnostic test                    development"],"prefix":"10.1128","volume":"13","author":[{"given":"Reetika","family":"Chaurasia","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03v76x132","id-type":"ROR","asserted-by":"publisher"}],"name":"Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine","place":["New Haven, USA"]},{"name":"Luna Bioscience Inc.","place":["New Haven, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Jacobs","sequence":"additional","affiliation":[{"name":"Luna Bioscience Inc.","place":["New Haven, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Tang","sequence":"additional","affiliation":[{"name":"Luna Bioscience Inc.","place":["New Haven, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Songyu","family":"Dong","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03v76x132","id-type":"ROR","asserted-by":"publisher"}],"name":"Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine","place":["New Haven, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8344-2004","authenticated-orcid":true,"given":"Joseph M.","family":"Vinetz","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03v76x132","id-type":"ROR","asserted-by":"publisher"}],"name":"Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine","place":["New Haven, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.56367\/OAG-043-11310"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.14.2.296-326.2001"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/s1473-3099(03)00830-2"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0003898"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0004122"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0227055"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.trstmh.2010.07.002"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2009.03.012"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2208"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-662-45059-8_5"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0004403"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1136\/bmj.1.5638.231"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cis035"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1017\/S0950268812001951"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0008074"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0129236"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.40.3.480-489.2002"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-03658-0"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0002468"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2022.1092197"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.859680"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.926994"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jprot.2024.105106"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","unstructured":"Giraud-Gatineau A Nieves C Harrison LB Benaroudj N Veyrier FJ Picardeau M. 2024. 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