MULTI-AFFINITY NANOTRAPS THAT ENHANCE DETECTION OF LOW-ABUNDANT PROTEINS: A NOVEL AND HIGHLY SENSITIVE TEST FOR THE DIAGNOSIS OF LYME DISEASE
Creators: name: MAGNI, RUBEN
Publisher: name: Universit� degli Studi di Milano
Year: 2017

description: In the recent years a lot of emphasis has been placed on the discovery and better detection of clinically relevant biomarkers. Biomarkers are crucial for the early detection of several diseases, and they play an important role in the improvement of current treatments, thus reducing patient mortality rate. Biofluids account for 60% of the human body mass and can be a goldmine of significant biomarkers. Unfortunately, low abundance biomarkers are difficult to detect with mass-spectrometry or immunoassays because of their low concentration in body fluids, their lability, and the presence of high-abundance proteins (i.e. albumin and immunoglobulins). In order to overcome these physiological barriers, we developed nanoparticles made of poly(N-isopropylacrylamide) (NIPAm) and functionalized with affinity reactive baits that one single step capture, concentrate and preserve labile biomarkers in complex body fluids (i.e. urine, blood, sweat, CSF). The design, synthesis and application of the Nanotrap hydrogel particles are described in this thesis. The novelty of the technology relies in the fact that in the past hydrogel nanoparticles have been studied and used as a drug delivery tool, whereas our application focuses on their capturing abilities instead of the releasing of specific drug molecules. Once the functionalized nanoparticles are incubated with a biological fluid, low molecular weight biomarkers are captured by the affinity baits while unwanted high abundance analytes are excluded. The potentially relevant biomarkers are then concentrated into small volumes and analyzed. The concentration factor (up to 10000 fold depending on the initial volume) enhances the effective sensitivity of mass-spectrometry and immunoassays and permits to detect previously invisible proteins thus improving biomarker discovery and diagnostic testing. This thesis discusses the use of hydrogel nanoparticles to develop a urinary antigen test for the detection of Lyme Borreliosis. 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 and to develop a diagnostic tool to monitor therapy success. Borrelia burgdorferi is the causative agent of Lyme disease. Using our hydrogel particles (Nanotraps) we evaluated the presence of 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. We employed Nanotraps 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. OspA 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&lt;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. 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 negative. Detection of OspA was performed using Western blot analysis. In order to obtain a quantitative measurement of the antigen, an ELISA was developed. Preliminary results showed a lowest limit of detection of 0.5pg/ml and %coefficient of variation 2%, dynamic range 0.5pg-30 pg/ml. 3/3 of symptomatic patients that resulted positive with the western blot Lyme assay were also found positive when tested on ELISA. Another promising format under development uses Mass Spectrometry Multiple Reaction Monitoring (MRM) for the detection of multiple Borrelia proteins after Nanotrap processing. Developing a diagnostic test against a panel of analytes will improve clinical sensitivity and understanding of staging of disease. MRM is a prime technology that yield multiplex measurement of more than 100 peptides in a single sample. A sensitivity of 5pg/ml and high reproducibility in human urine spiked with OspA was observed. Lastly, partially degradable Nanotraps were employed to produce a prototype of Lateral Flow Immunoassay (LFI) which exploits the use of antigen displaying nanoparticles for a point of care test for Lyme disease. This technology will ensure high accuracy and sensitivity while allowing for rapid testing of Lyme disease antigens in the urine of patients in the doctor office.
In summary, this study presents data supporting the successful use of the Nanotrap technology to develop a more accurate and sensitive test for Lyme disease that can diagnose the disease before seroconversion and that can be used to monitor therapy success. Nanotraps increase the effective analytical sensitivity of western blot analysis, ELISA, mass spectrometry MRM and lateral flow immunoassay. This is a concept that can be extended to communicable diseases with different etiologic agents (e.g. Tuberculosis, Chagas disease, Toxoplasmosis, etc.).