Development of a Sensitive Double-Antigen Sandwich ELISA for Human Papillomavirus 52 Subtype Antibody Detection Based on L1<sub>1-141</sub> Protein

Background: Human papillomavirus (HPV) subtype 52 is a high-risk genotype strongly associated with cervical carcinogenesis. Following persistent infection, HPV type 52 (HPV52) can induce dysregulated proliferation of cervical epithelial cells, ultimately contributing to malignant transformation. Given its oncogenic potential, early detection of HPV52 infection is critical for effective disease management and cervical cancer prevention. Objectives: To develop a novel double antigen sandwich enzyme-linked immunosorbent assay (DAgS-ELISA) for the determination of HPV infection status in patients initially using vaginal douche samples. Methods: This work employs truncated L1 protein (Aaa 1 - 141) as the detection antigen. The gene fragment encoding L11-141 protein was cloned into the pET32a vector, and the recombinant protein was expressed and purified. For enzyme-linked immunosorbent assay (ELISA), the L11-141 protein was coated onto plates at concentrations of 10 μg/mL and 20 μg/mL. Patient vaginal wash samples served as the antibody to be tested, while horseradish peroxidase (HRP)-conjugated L11-141 was used as the detection antigen at dilutions of 1:200 and 1:500. HPV antibodies in the samples were detected using a method with a cut-off (CO) value set at twice the negative control. The assay was validated using 115 clinical samples, including both negative and positive cases. Results: The optimized ELISA conditions included a coating antigen concentration of 20 μg/mL and a detection antigen dilution ratio of 1:200. Among the 115 clinical samples tested, 79 (68.7%) were positive, and 36 (31.3%) were negative. The method demonstrated a high sensitivity of 94% and specificity of 100%. Conclusions: This study using truncated L1 protein successfully established a DAgS-ELISA for direct detection of HPV antibodies in vaginal wash samples. Compared to traditional serological assays, this method offers several advantages: High sensitivity and specificity, minimal equipment requirements. Additionally, its simplicity and versatility make it suitable for large-scale primary screening in diverse settings.