AIM #3

Aim #3: Screen 100 bladder cancer (BC) and 100 normal samples using LFA to estimate sensitivity and specificity of the assay.

Samples: The BC patient samples will consist of various stages including both muscle non-invasive (Ta/T1) and muscle invasive (T2/T3) BC urine samples. Normal samples will be collected early morning (when concentration of biomarkers is highest) from age-matched group with no history of malignancy. De-identified urine from 100 consented BC patients in Dr. Ho’s clinic will be utilized in the study.  Dr. Ho will also identify and provide plasma from 100 patients with no history of malignancy. QSOX1-L in urine will be quantified by ELISA per protocol above and then compared to LFA results obtained with the same antibodies using similar capture/detector Ab configuration. A correlogram between ELISA and LFA will be generated to confirm the results. In addition, a Western Blot assay will be run on all samples to further confirm QSOX1-L identity. To establish a reference range for QSOX1-L levels in urine from patients and individuals without malignant disease, we will calculate the mean concentrations, ±2 SD.

Statistical analysis: To determine positive and negative predictive values, an empirical receiving operating characteristic (ROC) curve will be generated for QSOX1 ELISAs using a blind (re-coded) set of true positives and true negatives.  ROC (.20), the sensitivity of a QSOX1-based test with specificity 80%, will be estimated empirically, and the corresponding confidence interval will be calculated based on normal approximation to the asymptotic distribution of a logit transformed ROC function.  Area under the curve (AUC) and partial area under the curve (pAUC) on the interval [0, 0.2] will be estimated for the ROC curve with respective confidence intervals. In LFA, for each test line and/or ratio of test/control, the ROC curve will be calculated and plotted. The sensitivity, specificity, positive predictive value, and negative predictive value will be determined.

Milestone: Specificity and sensitivity of the ELISA and LF tests are determined.