Nanoglow substrate

The LIPSTICKS technology [24 (link)] 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.

The portable, handheld EnSURE photodiode luminometer (Hygiena) was also employed for testing. In order configure the device for LIPSTICKS, the Ultrasnap cap tubes supplied by the manufacturer (Hygiena) were first emptied of their contents and then rinsed four times with distilled water. The cleaned, empty tubes were then refilled with 100 μl Nanoglow substrate (Promega). To quantify the light emitted by the antigen/antibody complex, magnets bound with immune complexes were simply dropped into the Ultrasnap tube containing Nanoglow substrate, recapped and placed in the EnSURE luminometer and measured with an integration time of 15 s. Unlike Renilla luciferase antigen fusions, the NanoLuc reporter produced a high output with a stable glow with its substrate and is highly detectable during the long integration time with the handheld luminometer. Due to additional integration time of 15 s needed for the handheld luminometer, these tests required 1 min for completion.