Signal Enhanced HPV Protein Detection for Paper-based Dipstick Assays

Introduction:: Introduction: Cervical cancer has been the fourth most common cancer amongst women globally in 2020, where ~90 % of new cases and deaths occurred in low-and middle-income countries (LMIC) [1]. By 2030, the World Health Organization anticipates a 27% increase in cervical cancer mortality in LMIC compared to 1% increase in developed countries [2]. Persistent human papillomavirus (HPV) infections are primarily responsible for causing cervical cancer. Current HPV screening technologies cannot combine highly sensitive and specific detection within single-visits at the point of care (POC). Leveraging our lab’s expertise in paper-based diagnostic devices, we generate lateral flow immunoassays (LFIA) to detect protein biomarkers. Signal amplification using enzymatic substrates can be introduced to improve the test’s sensitivity by detecting samples at lower protein concentrations. In this case, the POC test targets HPV16 L1 capsidic proteins for early cervical cancer screening with collaborators in Zambia.




Materials and Methods:: The antigen-antibody binding for HPV L1 protein detection utilized Anti-HPV16 L1 antibodies (mouse monoclonal, Santa Cruz Biotechnology) conjugated with 40 nm gold nanoparticles (Cytodiagnostics) to HPV16 L1 recombinant proteins (Abcam). The pH-adjusted gold nanoparticles (AuNP) were incubated with the Anti-HPV L1 antibody (0.01 μg/mL, 30 mins, 25⁰C). For signal enhanced samples, streptavidin-poly HRP (9.7 μg/mL) was introduced before BSA and incubated within the AuNP-antibody solution for 30 minutes. Then, 10% BSA was added to block remaining binding sites on the AuNP surface. The conjugated AuNPs were then resuspended in 1% BSA and characterized using UV-Vis and dynamic light scattering (DLS). This solution was used for dot blot experiments and dipstick assay tests. A LFIA was designed in a dipstick format as seen in Figure 1, where the secondary IgG antibodies (Santa Cruz Biotechnology) were immobilized on the nitrocellulose detection membrane. Figure 1 also highlights the conjugated solution’s sandwich assay configuration before and after signal amplification using HRP-DAB.


Results, Conclusions, and Discussions:: The DLS data demonstrated the size increase after each conjugation step. The AuNP-antibody-BSA-L1 protein solution increased in size accordingly: 43.56, 49.72, and 61.79, and 69.54 d.nm as shown in Figure 2A. Figure 2B validates HRP conjugation to AuNP-antibody-BSA solutions at various detector antibody concentration. The conjugation protocol confirmed negligible aggregation. Conjugated samples were compared after one week to ensure stability. Dot blot and dipstick experiments identified protein detection as low as 150 ng/mL, well within the target clinical concentrations (50-200 ng/mL). Ongoing work utilizes HRP amplification to confirm a lower limit of HPV L1 detection at 50 ng/mL. To improve reproducibility, a XYZ3060 dispenser (Bio-Dot) will provide consistent dotting/stripping upon the detection pad. Also, two secondary capture antibodies and/or a control protein (β -actin) will be printed to represent a test and control line to confirm highly specific binding. Additional PBS washes and blocking reagents may be required to minimize the background signal upon the nitrocellulose membrane. With an increased sample size coupled with these improvements towards device fabrication and testing, this HPV screening tool can provide the cost-efficient and improved signal detection/sensitivity needed in low-resource settings.

Acknowledgements (Optional): : This work was funded in part by NIH NCI grant R01CA246315.


References (Optional): : [1] H. Sung, et.al., CA Cancer J Clin, 2021, vol.71, no.3, pg. 209-249.

[2] S. Krivacsy, et.al., Lancet Glob. Health, 2019, vol.7, no.12, e1605-6.