A time-resolved fluorescence immunoassay for rapid and precise automatic quality control of human papillomavirus type 68 VLPs in human papillomavirus vaccine.

The effectiveness and necessity of human papillomavirus (HPV) vaccination to prevent HPV infection and cervical cancer are increasingly recognized by people. The 15-valent HPV vaccine, which protects against almost high-risk types of HPV viruses identified by WHO, has attracted much attention. However, as the valence of vaccines increases, quality control in the HPV vaccine production process is facing more challenges. The precise quality control of the HPV type 68 virus-like particles (VLPs), one of the unique components of the 15-valent HPV vaccine that distinguishes it from existing vaccines, is the new requirement for vaccine manufacturers. Here we developed a novel time-resolved fluorescence immunoassay (TRFIA) for rapid and precise automatic quality control of HPV68 VLPs in HPV vaccine. Two murine monoclonal antibodies specifically targeting the HPV68 L1 protein were used to establish a classical sandwich assay. Except for pretreating the vaccine sample, the whole analysis process was performed by a fully automated machine, which saves detection time and gets rid of manual error. Multiple experiments established that the current novel TRFIA can efficiently and reliably analyses HPV68 VLPs. Present novel TRFIA has exhibited merits with speed, robustness, high sensitivity with a minimum detection value of 0.08 ng/mL, considerable accuracy, a wide detection range (up to 1000 ng/mL) and excellent specificity. It is also expected to provide a new detection method for quality control for each HPV type VLPs. To summarize, the novel TRFIA is of great interest for application in HPV vaccine quality control.

A custom-made time-resolved fluoroimmunoassay for the quantitation of the host cell protein of Vero in rabies vaccine.

Host cell proteins (HCPs) are the process-specific and inevitable impurities during the manufacture via a host cell, which affect the safety or efficacy of the bio-product. However, the commercial HCP enzyme-linked immunosorbent assay (ELISA) kits may not apply to specific products such as rabies vaccine from Vero cells. More advanced and process-specific assay methods are needed in the quality control of rabies vaccine throughout the whole manufacturing process. Therefore, a novel time-resolved fluoroimmunoassay (TRFIA) for the detection of process-specific HCP of Vero cells in rabies vaccine was established in this study. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was used during the preparation of HCP antigen. Based on a sandwich-type immunoassay format, analytes in samples were captured by one antibody coating in the wells and "sandwiched" by another antibody labeled with europium chelates. Due to the complex composition of HCP, both the capture and detected antibodies are polyclonal antibodies from the same anti-HCP antibodies pool. Multiple experiments have identified the optimal conditions to allow the valid and reliable detection of HCP in rabies vaccine. The TRFIA had a satisfactory limit of detection value (0.011 µg/ml) under optimal conditions, with the linear range from 0.0375 to 2.4 µg/ml of HCP. The coefficient variations (CVs) were all < 10%, and the recoveries were in the range of 97.00-102.42%. All the test results of Vero cell protein reference substance were included in the expected concentration, which demonstrated that the present method was available for the test of HCP in rabies vaccine. Based on these results, the novel TRFIA to detect HCP appears to be important for application in modern vaccine quality control during the whole manufacturing process.

Dual-color quantum dot-loaded nanoparticles based lateral flow biosensor for the simultaneous detection of gastric cancer markers in a single test line.

A dual-color quantum dots-loaded nanoparticles (QPs) based lateral flow biosensor with single test line has been developed. Red and green emitted QPs were conjugated with antibodies and served as detecting probes in assays respectively, while the mixture of various antibodies were immobilized on nitrocellulose membranes as one detection line. Benefit from eliminating the heterogeneity caused by different position on the membrane, current biosensor achieved higher accuracy comparing with prevalent multi-lines or multi-strips lateral flow systems, which is of great significance for analyzing ratio-related diagnostics. The capability and reliability of the multiplex biosensor are also demonstrated by utilizing pepsinogen I (PG I) and pepsinogen II (PG II) as the model analytes. Under the optimal conditions, quantitative detection was achieved with ultra-low limits of detection at 6.9 pM (0.29 ng mL-1, PG I) and 15.7 pM (0.66 ng mL-1, PG II) respectively. The spectra crosstalk was negligible and no apparent cross-reaction was found in simultaneous detection. Furthermore, a good linear correlation of the QPs based lateral flow biosensor and commercial time-resolved fluoroimmunoassay was obtained in the detection of clinical samples, indicating the high reliability of the proposed biosensor.