Luciferase-Based Detection of Antibodies for the Diagnosis of HPV-Associated Head and Neck Squamous Cell Carcinoma

Burbelo, Peter D.; Chaturvedi, Adrija; Notkins, Abner L.; Gunti, Sreenivasulu

Essentially as described, the LIPS assays were performed in approximately 2.5 h using a 96-well plate format at room temperature [19] [20] [21] . Briefly, recombinant luciferase antigen lysates were produced from transfection of DNA plasmids constructs into Cos1 cells and their activity in light units (LU) was determined with a tube luminometer (Turner Design 20/20). To initiate testing, 40 µL of buffer A, 10 µL of diluted human sera (1 µL equivalent), and the luciferase-antigen cell extract (input of approximately 10 million LU), diluted in buffer A, were added to each well of a microtiter plate for 1 h. Next, a 30% suspension (6 µL) of Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL, USA) was added to the bottom of each well of a 96-well filter HTS plate (Millipore, Bedford, MA, USA). The 100 µL antigen-antibody reaction mixture was moved to a filter plate and incubated for 1 h on a rotary shaker. Multiple washing steps of the retained protein A/G beads were then performed. After completion of the washing steps, LU were measured in a Berthold LB 960 Centro microplate luminometer (Berthold Technologies, Bad Wildbad, Germany) using either coelenterazine or furimazine substrate (Promega, Madison, WI, USA) for detecting Renilla luciferase and NanoLuc activity, respectively.