Our overall objective is to develop a low cost non-invasive Rapid Diagnostic Test (RDT) for the detection of BC from urine based on an enzyme biomarker QSOX1 overexpressed in most cancers 1, including bladder, with which we will test urine samples from control and test populations. Our hypothesis is that (i) QSOX1-L or QSOX1-related polypeptides can also be shed from BC urothelium into urine; (ii) patients with BC will show elevated levels of QSOX1-L in urine compared to patients with non-malignant diseases and (iii) QSOX1-L levels will decrease post-surgery and/or with medical treatment and rise with recurrence of tumor or progression of disease.

We will address our overall objective with the following specific three aims:

specific aims

Aim #1: Develop and validate at least five polyclonal rabbit antibodies targeting different epitopes of QSOX1-L isoform: Express and purify in E. coli expression system 100aa C-terminal polypeptide that differentiates QSOX1-L from QSOX1-S. Synthesize a set of four 12-18aa peptides covering specific regions of 100aa polypeptide. Immunize a set of animals with the five peptides conjugated to KLH. Validate the purified antibodies by ELISA and western blot using recombinant QSOX1-L.

Aim #2: Establish and validate sandwich ELISA and Rapid Diagnostic Test to quantify levels of QSOX-L in urine: Develop functional assay reagents based on the anti-C-terminal antibodies developed in Aim #1. Validate the reagents in a model system consisting of recombinant QSOX1-L. Evaluate initial functionality, limits of detection, and cross-reactivity.

Aim #3: Validation studies with blinded urine samples: Properly collected samples from Mayo Clinic Scottsdale Urology Department will be screened with the available antibodies in dot-blot, western blot, and ELISA formats to validate the performance of prototype RDT. The performance of the Phase I test (sensitivity, specificity) will be evaluated with over 100 BC negative and 100 BC positive preserved urine samples. 

At the conclusion of Phase I, we will have a set of at least four polyclonal antibodies specific to QSOX1-L isoform, a quantitative ELISA and a functional point-of-care prototype device capable of detecting QSOX1-L in urine. In Phase II, the RDT will be used to conduct clinical studies to prepare for FDA approval and commercialization. The final product will an easy to use and cost-efficient tool to diagnose BC non-invasively. Moreover, such a test could be used to follow patients’ post-surgical resection and monitor response to therapy. The results of this study will also form a foundation for the future applications of QSOX1-L biomarker for the detection of other tumors from associated proximal fluids or, possibly, as a pan-cancer biomarker detected from blood that circulates through all tumors and other tissues.