A Carboxyl-Coupled Light-Initiated Chemiluminescence Assay for HPV Bivalent Vaccine Potency

Background/Objectives: The development of human papillomavirus (HPV) vaccines is increasingly trending toward multivalency, and accurate quantification of HPV virus-like particles (VLPs) is critical for vaccine quality control. All marketed products currently rely on ELISA-based antigen quantification, which is complex and time-consuming. In accordance with the 3Rs principles, in vitro potency testing is expected to progressively replace in vivo testing. Therefore, efficient and convenient antigen quantification methods for multivalent HPV vaccines are essential to improve testing efficiency. Methods: In this study, photosensitive substances were loaded into beads of different diameters using a swelling method, and carboxyl groups were introduced onto the bead surfaces. By coupling HPV monoclonal antibodies via carboxyl groups, the use of high-risk coupling reagents was avoided, and a one-step light-initiated chemiluminescence assay (LiCA) was established for the in vitro relative potency testing of HPV vaccines. Results: The LiCA method established using 200 nm carboxyl-coupled beads for the HPV16/18 bivalent vaccine exhibited excellent linear range, specificity, accuracy, and precision. The LiCA results were comparable to those of ELISA; moreover, compared with ELISA, LiCA showed a lower detection limit and a wider dynamic range for both HPV16 and HPV18 VLP antigens, and was more convenient and time-saving to operate. Conclusions: A one-step LiCA was established using carboxyl-coupled beads with optimized diameters, providing an efficient and convenient approach for the in vitro relative potency testing of multivalent HPV vaccines based on large-particle VLP systems, which contributes to improved vaccine quality control and testing efficiency.