Fig 1.
Multiplex serotype-specific NS1 microcapillary immunoassay using stackable Cygnus devices.
(A) Reel of 0.5 Km of Microcapillary film. (B) Individual capillaries can be coated in bulk before being cut to 47 mm test strips. Duplicate capillaries were coated with serotype specific antibodies. Negative capillary was left uncoated and the positive capillary were coated with NS1 antigen. (C) Single gravity driven cassette with MCF filled with blood. (D) A block of 12 Cygnus cassettes clipped together with the end of the MCF located in the strip well. (E) A block of Cygnus cassettes interfaced with the custom stripwell. (F) Flow diagram of assay steps. (G) Individual antigens (25 ng/mL) were spiked into undiluted heparinised-plasma.
Table 1.
DENV NS1 antibodies used in the Cygnus test.
Fig 2.
Standard curves for Dengue NS1 detection by Cygnus devices, and examples of dengue patient samples.
Serial dilutions of recombinant NS1 of the indicated serotypes was spiked into (A) 4% BSA or (B) plasma and tested with multiplex MCF in Cygnus devices, n = 4, error bars indicate ± SEM. (C) A set of patient plasma samples were tested in batches of 6 or 12, and images of two example batches of 6 patient samples are shown. The dengue serotype determined by RT-PCR, conventional microplate ELISA and Cygnus are indicated underneath each sample.
Table 2.
Detection and quantitation limit of DENV NS1 by MCF in Cygnus devices.
Fig 3.
ROC curve analysis for DENV1-4 NS1 detection by MCF in Cygnus devices.
A receiver operator characteristic plot for each pair of NS1 capillaries was plotted against PCR determination for the full set of samples vs controls. The table below outlines the key performance indicators for the rapid multiplexed immunoassay devices with this clinical sample set.
Fig 4.
Comparison of detection performances among three NS1 assays.
Sensitivities (A) and specificities (B) of MCF (Cygnus), ELISA (5-pairs) and LFT are compared. The error bars show 95 percent confident intervals of the sensitivity and specificity of each assay. The difference in performances is compared pairwise using McNemar’s exact tests. Due to multiple comparisons, the threshold for statistical significance is adjusted to < 0.017 based on Bonferroni correction.
Table 3.
Overall performances for DENV diagnosis of NS1 Cygnus, NS1-microplate ELISA and NS1-LFT in comparison to standard RT-PCR.
Table 4.
Correlation of NS1-Cygnus performance in comparison with NS1-ELISA or NS1-LFT to detect NS1 in clinical specimens.
Table 5.
Serotyping performance of NS1-Cygnus compared to RT-PCR in 255 clinical samples.
Table 6.
Serotyping performance of NS1-microplate ELISA compared to RT-PCR in 255 clinical samples.