Poster Y3 - Developing a Point-of-Care Cervical Cancer Screening Test Based on Valosin-Containing Protein Detection

Introduction: Cervical cancer is the fourth most common cancer in women and the fourth leading cause of cancer-related deaths among them globally. Current cervical cancer screening techniques, including the Papanicolaou smear test and human papillomavirus DNA testing, are expensive and time-consuming, possess low sensitivity or specificity, and require trained personnel and sophisticated instrumentation. These drawbacks limit their efficient implementation in low-resource settings, where roughly 84% and 88% of cervical cancer cases and deaths take place every year, respectively. Therefore, a low-cost, rapid, sensitive, and specific point-of-care early-stage cervical cancer diagnostic assay is urgently needed. Valosin-containing protein (VCP) is innately involved in clearing misfolded proteins from the endoplasmic reticulum. In cancer cells, VCP aids in removing the abundant misfolded proteins from the cells, thereby facilitating their survival and further proliferation. Interestingly, higher expression of this protein has been identified in cervical cancer patients, making it a promising cervical pre-cancer and cancer protein biomarker. This study focuses on developing a point-of-care VCP lateral flow immunoassay (LFIA) for cervical cancer screening.

Materials and

Methods: Carrier-free rabbit monoclonal anti-VCP detector antibody (dAb – from Abcam) was conjugated to 40-nm gold nanoparticles (AuNPs – from Cytodiagnostics) via passive adsorption. Various dAb-to-AuNP molar ratios were evaluated using commercial quality control (QC) dipstick tests and dynamic light scattering (DLS) to select the conjugate with the highest signal intensity and stability. The mean grayscale intensity of the QC test’s anti-rabbit immunoglobulin G (IgG) line, to which the conjugates would bind, was quantified using the ImageJ software. The DLS stability parameters included the average hydrodynamic diameter and polydispersity index (PdI). The chosen conjugate was then used to determine the optimal goat anti-rabbit IgG capture antibody (cAb) concentration for the LFIA’s control line using in-house dipstick tests. The in-house assays were made by striping pre-cut nitrocellulose membranes with the anti-rabbit IgG using the BioDot XYZ3060™ dispense system. After further membrane blocking, washing, and drying steps, the tests were assembled and run with the optimal conjugate to evaluate whether the striped cAb produced sufficient signal.

Results, Conclusions, and Discussions: Of the studied dAb-to-AuNP molar ratios, 35:1 had the highest grayscale intensity (27.17 ± 1.22 – Figure 1A and B) with a diameter and acceptable PdI of 100.80 ± 0.51 nm and 0.25, respectively. This conjugate was also the most stable both intensity- and size-wise throughout a 7-day stability study. Hence, it was chosen as the optimal conjugate for the LFIA device. Furthermore, the striped 1.5 mg/mL anti-rabbit IgG generated the anticipated high colorimetric signal (21.02 ± 0.42), and was therefore, selected as the optimal concentration for the LFIA control line (Figure 1C). The next steps will determine the test line’s anti-VCP cAb concentration using the in-house dipstick assays and the optimized parameters will be incorporated into the VCP LFIA design. The device performance will be evaluated by testing it with lysates of cultured cervical cancer cells and clinical cervical swab specimens. This low-cost rapid test will enable sensitive and specific cervical cancer screening in resource-limited regions, leading to a more timely diagnosis and, ultimately, more effective treatment. Of note, VCP may also be multiplexed with other key cervical cancer proteins, allowing for a more reliable identification of these lesions.

Acknowledgements (Optional): This project is funded by NIH R01CA246315.