The E6 strip test is based on detection of HPV-E6 oncoprotein in cervical swab samples. The current prototype detects E6 oncoprotein of HPV type 16, type 18, and/or type 45 on three distinct test lines.
Briefly, the Dacron swab collection tip is placed in 0.933 ml of extraction buffer for 30 min with constant mixing. Then, 87 μl of proprietary buffer was added, and the mixture was incubated with mixing for another 30 min. The extracted, lysed specimen was clarified by centrifugation in a microcentrifuge at 13,000 × g for 10 min.
An aliquot of the specimen lysate (0.12 ml) was combined with a detector monoclonal antibody (MAb) cocktail consisting of two anti-E6 MAbs, one that binds to HPV16 E6 and another that binds to HPV18 E6 and HPV45 E6. Upon placement of the HPV E6 strip test into the vial containing the specimen lysate and alkaline phosphatase (AP)-conjugated detector MAbs, this mixture (referred to as test solution) migrated up the nitrocellulose membrane of the E6 strip test, driven by capillary forces. Capture of the E6 oncoprotein-detector MAb complex occurred using monoclonal anti-E6 oncoprotein antibodies (capture MAbs) immobilized onto the nitrocellulose membrane of the E6 strip test as three distinct “test lines.” Each test line consists of one capture MAb specific for HPV16 E6, HPV18 E6, or HPV45 E6. Conjugation of the detector MAbs with AP allowed for visualization of immobilized detector MAb via a colorimetric reaction with AP substrate, resulting in a red-purple deposit. A fourth line, located above the three test lines consisted of goat anti-mouse Ig antibody and served as an internal control for proper test solution flow and for proper function of the detection system. Test solution migration via the lateral-flow strip occurred over approximately 56 min. Next, the lateral-flow strip was placed into a small volume (0.05 ml) of wash solution for two 6-min wash steps. Finally, the lateral-flow strip was submerged into a vial with AP substrate solution, and the colorimetric reaction was developed for 15 min. After development and removal of the lateral-flow strip from the AP substrate vial, test outcome was obtained via visual inspection.
Aliquots of specimen lysates were also tested for 37 HPV genotypes as previously described (2
). HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 were defined as the carcinogenic HPV genotypes (1