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

The LIPSTICKS technology [24] employed cell extract containing NanoLuc-E6 protein was investigated for ultrarapid diagnosis of HPV-associated HNSCC. Compared to the previously reported protocol, a slightly modified version of the assay was used which changed the order of reagent addition and produced a higher signal. To perform the modified version of the assay, 5 µL of the diluted serum sample (1:50 in water) is added to 5 µL of the NanoLuc-E6 fusion protein cell extract (30 million LU/ µL) in a 1.5 mL microfuge tube and then 5 µL of diluted paramagnetic beads (Thermo Scientific/Pierce ® protein A/G magnetic beads, Waltham, MA, USA), diluted 1:5 in water are added. The reaction mix is tapped two times to disperse the magnetic beads and then 100 µL of buffer A is pipetted into the reaction mixture and the tube is immediately vortexed for 2 s. A 1/8 diameter neodymium magnetic stick (K & G Magnets, Pipersville, PA, USA) is then immersed into the tube containing the beads for 5 s to collect the immune complexes. The magnet is removed and dipped twice in wash buffer A. Lastly, the magnetic stick is placed in a tube, preloaded with 100 µL of the Nanoglow substrate (Promega) and the luminescent glow is measured with the tube luminometer with an integration time of 1 s.