Desk S4. epitope to a small particular OspA C-terminal domain name OspA236-239 conserved across infectious species but with no homology to human proteins and no cross-reactivity with relevant viral and non-bacterial proteins. 268 urine samples from patients being evaluated for all those categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to end result, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. Results OspA test characteristics: sensitivity 1.7?pg/mL (least expensive limit of detection), % coefficient of variance (CV)?=?8?%, dynamic range 1.7C30?pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p? ?10?6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology end result was 87.5?% (21 urinary OspA positive/24 serology positive, Chi squared p?=?4.072e?15). 41 of 100 patients under surveillance for prolonged LB in an endemic area were positive for urinary OspA protein. Conclusions OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA unfavorable. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0701-z) contains supplementary material, which is available to authorized users. Background Prompt antibiotic treatment of early stage Lyme borreliosis (LB) can prevent progression of the disease from your localized stage to the early and late disseminated stages [1, 2]. Regrettably, because the clinical presentation can be so varied, early stage disease can be misdiagnosed for a variety of reasons including failure to develop an Cariporide erythema migrans (EM) rash [3C6], failure of the patient or clinician to recognize an EM rash, if present [4C7], the non-specific nature of early symptoms (fatigue, fever, headache, muscle and Rabbit Polyclonal to RPC5 joint pains, swollen lymph nodes), and a negative, or ambiguous serology [8C12]. Moreover, even after a single first course of antibiotic therapy, a small but significant percentage of patients and experimentally infected animals can continue to harbor [13C15]. Thus there is a clinical need to improve the diagnostic specificity of early stage Lyme assays, particularly in the period prior to the mounting of a strong serologic response [8, 10]. In addition, it would be valuable to know with greater certainty whether a first round of therapy is successful or should be repeated because of persistence [8, 10, 16, 17]. To address these requires we evaluated urinary Outer surface protein A (OspA) in early stage LB using an analyte harvesting nanotechnology, Nanotrap particles, to achieve high sensitivity [18, 19], coupled with an anti-OspA monoclonal antibody (mAb) which we show herein to recognize a narrow specific OspA C-terminal region, OspA236-239. OspA26-239 sequence is usually conserved across infectious species, but does not have sequence homology with human or non-relevant pathogens. We selected OspA for urinary monitoring of early stage LB for several reasons including its central role in the early stage of pathogenesis [20], the known shedding of antigen in the urine of animals infected with (Bb) [21], and the OspA sequence conservation across species [22, 23]. OspA is usually a 273 amino acid protein that folds in an elongated conformation spanning 80 ? from end Cariporide to end. OspA binds to the surface of the spirochete at the N-terminus via a lipid anchor. The structure consists of 21 consecutive antiparallel -strands followed by a short -helix in the C-terminus and can be divided into two discrete domains: a sandwich domain at the N-terminus and a barrel domain at the C-terminus [23]. The OspA barrel domain name at the C-terminus is usually highly conserved across pathogenic species and plays an important role in Bb induced Cariporide immune tolerance, induction of the inflammatory response through TLR2 [14, 24], and host immunologic acknowledgement [20]. In this study we focused on the shedding of urinary OspA peptides that contain the crucial C-terminus domain name. Despite the strong rationale for evaluating antigens in body fluids for diagnostic purposes in patients with LB, including cerebrospinal fluid and urine [25, 26], the validity of previous immunoassays for proteins has remained controversial [27], due to questions of specificity and sensitivity. Previous immunoassays for antigens have employed polyclonal antibodies raised against.