Duplex Vertical-Flow Rapid Tests for Point-of-Care Detection of Anti-dsDNA and Anti-Nuclear Autoantibodies

Abstract

The goal of this study is to develop a rapid diagnostic test for rheumatic disease and systemic lupus erythematosus (SLE) screening. A novel rapid vertical flow assay (VFA) was engineered and used to assay anti-nuclear (ANA) and anti-dsDNA (αDNA) autoantibodies from systemic lupus erythematosus (SLE) patients and healthy controls (HCs). Observer scores and absolute signal intensities from the VFA were validated via ELISA. The rapid point-of-care VFA test that was engineered demonstrated a limit of detection of 0.5 IU/mL for ANA and αDNA autoantibodies in human plasma with an inter-operator CV of 19% for ANA and 12% for αDNA. Storage stability was verified over a three-month period. When testing anti-dsDNA and ANA levels in SLE and HC serum samples, the duplex VFA revealed 95% sensitivity, 72% specificity and an 84% ROC AUC value in discriminating disease groups, comparable to the gold standard, ELISA. The rapid αDNA/ANA duplex VFA can potentially be used in primary care clinics for evaluating patients or at-risk subjects for rheumatic diseases and for planning follow-up testing. Given its low cost, ease, and rapid turnaround, it can also be used to assess SLE prevalence estimates.

Keywords: anti-dsDNA antibody; anti-nuclear antibody; gold nanoparticles; rheumatic disease; vertical-flow assay.

Figure 1

The general principle of the VFA setup. dsDNA and Hep-2 cell nuclear antigens are spotted on the left as test zones, and biotinylated BSA at low and high concentrations are spotted on the right of the nitrocellulose membrane (NCM) as part of control zones. After wetting the NCM, the sample containing anti-dsDNA and/or ANAs is loaded for them to bind to their respective capture antigen substrate on the NCM, thus forming an antigen–antibody immuno-complex. Biotinylated anti-human IgG is then added for it to bind with the anti-dsDNA or ANAs. Finally, streptavidin-conjugated GNPs are added for them to bind with anti-IgG in test zones and with biotinylated BSA in control zones, displaying a green colorimetric signal on the NCM. Between each step, the membrane is washed to remove unbound materials and to prevent nonspecific binding.

Figure 2

Serial dilution testing using the duplex ANA–αDNA VFA. (A) Image of serial dilutions of the positive control standard (ANA+ and αDNA), ranging from 10 IU/mL to 0 IU/mL, applied to the duplex ANA–αDNA VFA in duplicate (top and bottom). (B) Dose–response curve created using a human anti-dsDNA positive control standard (ANA+ and αDNA+); plotted is the intensity observed in the ANA test zone (Hep-2, orange dashed line) and αDNA test zone (DNA, blue dashed line) versus the actual concentration applied. Colorimetric intensity values were calculated using ImageJ 1.52a. The LoD was determined to be the lowest assayed concentration, the intensity of which exceeded the threshold of the sum of the mean intensity of the non-spiked buffer (0 IU/mL) and 3× its standard deviation.

Figure 3

Assessing the storage stability of the duplex ANA–αDNA VFA. Images shown pertain to four sets of standard curves generated using duplex ANA–αDNA VFAs that were stored at room temperature for one day (A), one month (B), two months (C), and three months (D) after the immobilization of the antigen on the NCM, in duplicate. Serially diluted human anti-dsDNA positive control standard (ANA+ and αDNA+) from 10 IU/mL, 5 IU/mL, 1 IU/mL, and 0.5 IU/mL to 0 IU/mL were loaded onto the NCM to construct the standard curves. (E) Plots of serial dilution of the anti-dsDNA positive control standard (ANA+ and αDNA+) under different storage durations. Orange line indicates ANA and blue line indicates αDNA detection. The LoD of the four sets of standard curves was 0.5 IU/mL for ANA and αDNA detection, and there was no significant difference between the storage times among LoDs and linearities as assessed via one-way ANOVA. The LoD was determined to be the lowest assayed concentration, the intensity of which exceeded the threshold of the sum of the mean intensity of the non-spiked sample (0 IU/mL) and 3× its standard deviation.

Figure 4

Disease-discriminating power of the duplex ANA–αDNA VFA and ELISA. (A) Serum autoantibody levels in 22 SLE and 18 HC serum samples assayed using the duplex ANA–αDNA VFA (OS or IS) and ELISA. Their ability to discriminate SLE patients from HCs was assessed using ROC AUC values. (B–F) The levels of autoantibodies assayed in SLE patients and HCs using the 2 different platforms plotted and analyzed using a Mann–Whitney U-test. (G,H) SLEDAI and rSLEDAI disease activity values (Y-axis) in all SLE patients with these values (20 out of 22) plotted against αDNA levels (X-axis), as assayed via the VFA (IS or OS), with Pearson’s correlation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as determined using Mann–Whitney U-test. AUC: area under the curve; IS: imaging score; OS: observer score; SLEDAI: Systemic Lupus Erythematosus Disease Activity Index; rSLEDAI: renal SLEDAI.