Field Test and Validation of the Multiplier Measles, Mumps, Rubella, and Varicella-Zoster Multiplexed Assay System in the Democratic Republic of the Congo by Using Dried Blood Spots.

Here we describe baseline validation studies and field performance of a research-use-only chemiluminescent multiplex serology panel for measles, mumps, rubella, and varicella-zoster virus used with dried blood spots in support of the 2013-2014 Democratic Republic of the Congo Demographic and Health Survey. Characterization of the panel using U.S. FDA-cleared commercial kits shows good concordance for measles, mumps, rubella, and varicella-zoster with average sensitivity across assays of 94.9% and an average specificity of 91.4%. As expected, performance versus available standards validated for plaque-reduction assays does not provide a 1:1 correspondence with international units and yet demonstrates excellent linearity (average Hill's slope = 1.02) and ∼4 logs of dynamic range. In addition, for the four assays, the multiplexed format allowed for inclusion of three positive and two negative controls for each sample. A prototype Dynex Multiplier chemiluminescent automated immunoassay instrument with a charge-coupled device camera provided a rugged and robust processing and data acquisition platform. Performance of a multiplex instrument for serological testing in a substantially resource-limited environment shows excellent reproducibility, minimal cross-reactivity, and a clear discrimination between specific assays and should be considered a viable option for future serosurveys.IMPORTANCE The critical evaluation of immunization programs is key to identifying areas of suboptimal vaccination coverage, monitoring activities, and aiding development of public health policy. For evaluation of vaccine effectiveness, direct antibody binding assay methods, including enzyme immunoassay, enzyme-linked fluorescence assays, and indirect immunofluorescence assay, are most commonly used for detection of IgG antibodies. However, despite their well-demonstrated, reliable performance, they can be labor-intensive and time-consuming and require separate assays for each individual marker. This necessitates increased sample volumes, processing time, and personnel, which may limit assessment to a few key targets in resource-limited settings, that is, low- and middle-income locations where funding for public health or general infrastructure that directly impacts public health is restricted, limiting access to equipment, infrastructure, and trained personnel. One solution is a multiplexed immunoassay, which allows for the detection of multiple analytes in a single reaction for increased efficiency and rapid surveillance of infectious diseases in limited-resource settings. Thus, the scope of the project precluded a full validation, and here we present abbreviated validation studies demonstrating adequate sensitivity, specificity, and reproducibility.

Dynex: multiplex ELISA technology.

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.