Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

Abstract

ObjectivesPrompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB.MethodWe employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation.ResultsOspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7–30 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 outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e−15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.ConclusionsOspA 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 negative.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0701-z) contains supplementary material, which is available to authorized users.

Current state of the art for enhancing urine biomarker discovery

“…Extensive reviews of the translational research progress in the urinary proteomic field have been published [2, 41, 49-51]. Potential urinary biomarkers are being discovered in a variety of non-kidney associated diseases including acute appendicitis [52, 53], infectious diseases such as Tuberculosis [54], Chagas Disease and Lyme Disease [55-57], cancer [49, 50, 58-61], cardiovascular disease [62-64], and aging [65, 66]. Selected examples of the progress in urinary protein identification are described below, highlighting a) improvements in mass spectrometry-based urinary proteomic discovery, b) the number of identifiable proteins, and c) pathophysiologic associations over the last decade.…”

“…Rather than inventing new, more sensitive instruments, alternative approaches for detecting low abundance proteins rely on devising technologies for subtracting interfering high abundance proteins, enriching the low abundance protein fraction, or equalizing the abundance of both high and low abundance proteins. Two techniques for enriching low abundance proteins highlighted in this review are combinatorial peptide ligand libraries to enrich the low abundance proteins (CPLL, ProteoMiner™) (Figure 2) [22, 24, 104, 123, 127-130] and hydrogel nanoparticles (NP) for excluding high molecular weight proteins and simultaneously harvesting specific classes of proteins (Figure 3) (Table 3) [23, 57, 107, 131, 132]. …”

“…Lyme Disease is transmitted via ticks infected with Borrelia spirochetes, commonly B. burgdorferi in the eastern United States. Diagnosis is typically made based on the presence of erythema migrans (bulls eye) rash and/or positive serologic test for antibodies to Borrelia antigens [57]. Lyme Disease can be difficult to diagnose in the early stages due to vague or overlapping symptoms, lack of the classic erythema migrans rash, or indeterminate serology results, thus potentially delaying treatment.…”

“…NIPAm-acrylic acid nanoparticles containing the nondisperse dye Acid Black 48 were shown to sequester and concentrate B. burgdorferi outer surface proteins in urine, as well as from an individual Ixodes scapularis tick [56]. A non-invasive urine test to detect the presence of the B. burgdorferi antigens prior to development of antibodies could transform early diagnosis and treatment for Lyme Disease [57]. …”

“…Following the proof-of-principle study, the dye bait was optimized for concentrating Borrelia antigens [57]. NP containing the anthraquinone dye Remazol Brilliant Blue were used to enhance the sensitivity of detection of B. burgdorferi via western blotting with a monoclonal antibody against a specific C-terminal OspA domain [57].…”