Background: Treponema pallidum (Tp) is the causative agent of the multi-stage disease syphilis. Existing serology-based diagnostic tests have suboptimal sensitivity and specificity in early and late disease stages, are inadequate for diagnosing congenital syphilis, and are unable to differentiate current versus previous infection. Herein, we describe the development of novel immunoassays for detection of Tp proteins/peptides in easy-toobtain patient samples, including urine and plasma. Methods: Affinity purified polyclonal antibodies were developed against unique Tp biomarker peptides that had either been experimentally detected in patient samples, bioinformatically predicted to be present in patient samples, or derived from highly abundant Tp proteins. The automated Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) workflow was used to measure a panel of biomarker peptides from clinically confirmed syphilis patient samples. Briefly, this methodology involves enrichment of proteotypic endogenous peptides from trypsin-digested patient samples using high affinity antibody reagents followed by their identification by tandem-mass spectrometry. Current investigations are focussed on: (1) deriving monoclonal antibodies against each biomarker peptide to increase clinical sensitivity and ensure optimal assay performance and (2) developing parallel immuno-mass spectrometrybased assays and inexpensive, easy-to-use immunoassay-based tests. Results: To date, using polyclonal antibodies raised against individual biomarker peptides, the SISCAPAbased assay has detected Tp biomarker peptides in 80% of patient samples from individuals with clinically confirmed primary, secondary, latent and tertiary disease. Multiplexing the panel of polyclonal antibodies specific for the identified biomarker peptides increased the detection efficiency, with endogenous peptides detected in 90% of tested patient samples from all disease stages. Conclusion: The SISCAPA-based direct diagnostic assay was able to detect biomarker peptides in the majority of samples from patients with clinically confirmed syphilis from all disease stages, demonstrating proof-of-concept. Assay optimization via generation of monoclonal antibodies is currently underway, with the goal of enhancing sensitivity and generating a continual source of these mono-specific diagnostic tools. Development of this assay into both an immuno-mass spectrometry-based assay and an antigen capture enzyme-linked immunosorbent assay (ELISA)-based test will ensure worldwide reach of these simple and sensitive diagnostic immunoassays and definitive detection of active infectious and congenital syphilis cases. [ABSTRACT FROM AUTHOR]