Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus

Abstract

Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4°C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas.

Keywords: Diagnostic; ELISA; IgM; Kit; Yellow fever virus.

Figure 1

Stability of the YF MAC-HD kit over time. The stability of the kit was measured at 0, 3, 6 and 12 months after production and storage at either RT or 4 °C using 8 serum samples, where sample 8 was a background responder (not plotted). A 2 week stress test at 37 °C was performed on kits after 3 months of storage. Test results were reported as P/N values and were plotted alongside those of the Comparative MAC-ELISA.

Figure 2

Stability of the YF MAC-HD expressed as optical densities over time for kits stored at 4 °C and RT. Sample 8 was a background responder and was not plotted.

Figure 3

Receiver operator characteristic curves to determine P/N cut-off values in the YF MAC-HD and the YF MAC-ON. The optimal balance between sensitivity and specificity based upon the results with true positives and negatives was P/N of 1.6 for YF MAC-HD and 2.0 for YF MAC-ON.