The human papillomavirus (HPV) is the main cause of cervical cancer, being the fourth most common cancer in women worldwide. Fourteen HPV genotypes (HPV16, −18, −31, −33, −35, −39, −45, −51, −52, −56, −58, −59, −66 and − 68) are considered high-risk for cervical cancer. Almost 95 to 99 % of cervical cancer cases are associated with genital infections due to high-risk HPVs (hrHPV), with 70 % attributed to HPV16 and HPV18 (Derbie et al., 2020; Shrestha et al., 2018). Consequently, the HPV DNA test is commonly used for screening. However, the final diagnosis relies on colposcopy with cytology and/or immunohistochemistry, as HPV DNA testing has a high negative predictive value. This is primarily because the presence of HPV DNA may indicate an acute or transient infection rather than an established one. While most sexually active females contract HPV at least once in their lifetime, fewer than 10 % of them remain persistently infected and subsequently develop cervical cancer (Choi and Park, 2016). Conversely, cytology offers better specificity and is thus more suitable for diagnosis.

Cervical cancer is characterized by abnormal, uncontrolled proliferation of cells due to genetic and epigenetic changes that regulate their growth, differentiation and death (Dasari et al., 2015). The interaction of certain proteins, such as E6 and E7 HPV DNA in the host cell, is known to disturb the cell cycle. This results in an abnormal expression of protein biomarkers, including p16INK4a (also known as p16) and Ki-67. p16, a cyclin-dependent kinase (CDK) inhibitor, belongs to the tumor suppressor pathway that is deregulated in most human tumors (Romagosa et al., 2011). Ki-67, on the other hand, is frequently used as an indicator for cell proliferation in diagnostic and prognostic tools (Li et al., 2015; Menon et al., 2019). Its expression is significantly higher in malignant cells, ranging from mild dysplasia, known as low-grade intraepithelial lesion (LSIL), to moderate or severe dysplasia, called high-grade intraepithelial lesion (HSIL).

Protein biomarkers can provide clinicians with a better understanding of the disease and potentially permanent cell changes. An observational study conducted at Kaiser Permanente Northern California on HPV-positive women demonstrated that women with a positive p16/Ki-67 dual stain cytology should undergo immediate colposcopy, while those with negative dual-stain cytology can be monitored for up to two years (Wentzensen et al., 2015). Another meta-analysis concluded that p16 staining and p16/Ki-67 dual staining, compared with hrHPV DNA testing, improve the specificity of clinical care diagnosis (Peeters et al., 2019). Additionally, it was found that dual staining has a sensitivity comparable to that of HPV testing, whereas p16 staining alone failed to exhibit a similar efficacy (Bergeron et al., 2015).

The CINtec® PLUS assay, a test utilizing dual-biomarker technology to simultaneously detect the p16 and Ki-67 presence, has demonstrated higher sensitivity compared to cytology, with equal specificity, in triaging women with a history of LSIL (Ratnam et al., 2021). When compared with HPV testing, CINtec exhibited higher specificity but comparable sensitivity for detecting CIN2+ in women with LSIL cytology. In another study, it was demonstrated that the hrHPV test effectively triages women with atypical squamous cells of undetermined significance (ASC-US), identifying those requiring referral. However, it exhibited low specificity for those with LSIL (Peeters et al., 2019). Overexpression of p16, with or without Ki-67, has been reported to more accurately predict underlying cervical intraepithelial neoplasia grade 3 or worse (CIN3+) by indicating the neoplastic transformation of HPV-infected cervical cells.

A quantitative enzyme-linked immunosorbent assay (ELISA) shows promise as an alternative to current qualitative dual-stain immunohistochemistry tests. It eliminates the need for trained pathologists and is unaffected by the anatomical origin of the sample, allowing assessment of samples collected by gynecologists or patients themselves, based on preference or available infrastructure. Moreover, ELISA yields faster results at a lower cost, both in terms of reagents and required infrastructure. Implementing ELISA in routine screening can lead to more accurate decisions for follow-up visits in HPV DNA-positive women.

While limited attempts have been made to use ELISA, particularly for p16 in cervical cancer screening, no cutoff has been established for this application (Mao et al., 2007; Balasubramanian et al., 2009; Jentschke et al., 2013; Leung et al., 2021). These tests fall short, especially when testing self-collected samples, which are increasingly common. Considering these limitations, this article presents an optimized protocol for developing an ELISA assay to simultaneously measure p16 and Ki-67 in cervical samples, regardless of whether collected by clinicians or patients themselves. A dedicated sample preparation step is also developed to ensure the stability of target analytes during the measurement process.

The optimized ELISA protocol is then tested using different collection media commonly used to preserve clinical samples. For each medium, a calibration curve is obtained, determining the dynamic range, limit of detection (LoD) and limit of quantification (LoQ). Additionally, the optimized assay is validated on a set of real samples.