Advancing cervical cancer screening: A novel in-house ELISA assay for the simultaneous detection of p16 and Ki-67.

Immunostaining and cytology, commonly used to detect cervical cancer, are time-consuming and pathologist dependent. In this work, for the first time, we have developed an ELISA assay for the extraction and co-measurement of p16 and Ki-67 in both vaginal and cervical samples for cervical cancer screening. Novel custom-made antibodies were developed, and a competitive ELISA assay was developed and optimized based on the concentration and incubation time of different steps to improve the sensitivity and specificity of the final assay in assessing cervical samples. The as-developed ELISA assay shows high sensitivity and selectivity in different collection media. The LoD of the test is 0.00004 and 0.0058 μg/mL for p16 and Ki-67, respectively. The as-developed assay can be used in future studies to determine protein thresholds for different lesion grades of cervical cancer for samples collected in different settings. These assays can revolutionize the cervical cancer screening management process in the near future.