The analytic performance of a low-cost, research-stage DNA test for the most carcinogenic human papillomavirus (HPV) genotypes (HPV16, HPV18, and HPV45) in aggregate was evaluated among carcinogenic HPV-positive women, which might be used to decide who needs immediate colposcopy in low-resource settings (“triage test”). We found that HPV16/18/45 test agreed well with two DNA tests, a GP5+/6+ genotyping assay (Kappa = 0.77) and a quantitative PCR assay (at a cutpoint of 5000 viral copies) (Kappa = 0.87). DNA sequencing on a subset of 16 HPV16/18/45 positive and 16 HPV16/18/45 negative verified the analytic specificity of the research test. It is concluded that the HPV16/18/45 assay is a promising triage test with a minimum detection of approximately 5,000 viral copies, the clinically relevant threshold.
Multiplex assays have advantages in being able to reduce time and sample volumes; however, multiplexing offers additional challenges, which include developing multiple assays that can be run with the same protocol, reagents, and sample dilution. Moreover, transferring reagents that work in one assay format to another without compromising performance and the integrity of sample measurement is difficult. The U-PLEX® platform enables flexible multiplexing of immunoassays using MSD’s MULTI-ARRAY® technology.
Antibodies used in V-PLEX® kits were transferred to the U-PLEX platform by biotinylating the capture antibodies. The assays represented a variety of analyte classes (chemokine, interleukin, interferon), antibody types (monoclonal, polyclonal), and analytical properties (sensitivity, dynamic range, concentration-response slope). During development, antibody concentration, biotin and MSD SULFO-TAG™ label ratios, and calibrator concentrations were evaluated and optimized.
Assays were readily transferred to the U-PLEX platform with calibration curves showing expected signals, sensitivity, precision, and accuracy. Controls for the assays showed CVs of <10% within runs. Sensitivities were <1 pg/mL for many assays. All assays used the same assay diluents. Non-specific binding between assays was typically <0.1%. We measured 40 human serum and 40 EDTA plasma samples, and demonstrated good correlation with V-PLEX assays (r2 > 0.9; slopes 0.8 – 1.2).
The utility and convenience of the U-PLEX platform was demonstrated by easily transferring over 40 human cytokine assays onto the platform without compromising performance. When multiplexed, these assays enable multiple measurements from small amounts of human samples.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.