Development of a Bead-Based Multiplex Assay for Use in Multianalyte Screening and Surveillance of HIV, Viral Hepatitis, Syphilis, and Herpes

Abstract

Diagnostic assays that can simultaneously determine the presence of infection with multiple pathogens are key for diagnosis and surveillance. Current multiplex diagnostic assays are complex and often have limited availability. We developed a simple, multianalyte, pathogen detection assay for screening and serosurveillance using the Luminex Magpix platform that is high throughput and can be helpful in monitoring multiple diseases. The Luminex bead-based 10-plex immunoassay for the detection of HIV-1, HIV-2, Treponema pallidum, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), and HSV-2 infections was accomplished by coupling beads with specific antigens to detect IgG antibodies in plasma or serum samples. Each coupled antigen was systematically optimized, and the performance was evaluated using a panel of well-characterized specimens (n = 417) that contained antibodies to HIV-1, HIV-2, T. pallidum, HBV, HCV, HSV-1, and HSV-2. The multiplex assay had a sensitivity of 92.2% (95% Clopper-Pearson confidence interval [CI], 90.2 to 94.0%) and a specificity of 98.1% (95% CI, 97.6 to 98.7%). The sensitivities and specificities for disease-specific biomarker detection ranged from 68.7 to 100% and 95.6 to 100%, respectively. The results showed that the 10-plex immunoassay had an overall agreement of 96.7% (95% CI, 96.7 to 97.3%) with reference tests and a corresponding kappa value of 0.91 (95% CI, 0.90 to 0.93). Kappa values for the individual pathogens ranged from 0.69 to 1.00. The assay is robust and allows the simultaneous detection of antibodies to multiple antigens using a small sample volume in a high-throughput format. This assay has the potential to simplify disease surveillance by providing an alternative to expensive and highly specialized individual tests.

Keywords: blood-borne; multipathogen; multiplex.

FIG 1

Dot plot showing the distribution and antibody reactivity levels of the 10-plex antigens. The horizontal lines (black) show the cutoffs that separate the two groups within each parameter, and the middle horizontal line within each group indicates the mean value for the group (n = 417). Note that background noise (MFI from beads only) was not subtracted as its contribution was minimal to the overall MFI.

FIG 2

Concordance plot (A) and Bland-Altman analysis (B) between the monoplex assay and the multiplex assay. (A) MFI values were obtained from an analysis of a mixture of HIV-negative and HIV-positive specimens and analyzed for HIV reactivity in the monoplex (HIV antigens only) and multiplex (HIV antigens and all other antigens) formats. The line of best fit is represented by the dotted blue line with the statistics shown. (B) Bland-Altman plot comparing the monoplex to multiplex results on a log scale (n = 850).