Research ArticleDiagnostics

Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR

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Science Translational Medicine  07 Apr 2021:
Vol. 13, Issue 588, eabd9696
DOI: 10.1126/scitranslmed.abd9696
  • Fig. 1 sGP as a diagnostic target and selection of capture and detection Abs for the EBOV D4 assay.

    (A) Schematic of EBOV infection. (B) Schematic of the GP gene transcription profile. (C) Binding of scFv-phage clones to EBOV sGP measured using Ag-down ELISA. Each data point is N = 1 technical replicate for scFv-phage clones with a distinct sequence. a.u., arbitrary units. (D) Binding of six scFv-Fc Abs to sGP, measured using Ag-down ELISA. Each data point represents the mean ± SEM of N = 2 replicates. (E) Schematic of Ab pairing using the D4 assay. (i) cAb and FL-dAb printed onto POEGMA-coated glass are exposed to sGP-spiked sample. (ii) Fluorescent spots are imaged; insets show spots with high (white) and low (red) fluorescence output. (iii) LoD and DR of each pair are calculated. (F to H) Dose-response curves using all six scFv-Fc Abs as cAb and C2BA5-2 (F), C2B6-2 (G), and A1F3-1 (H) as the dAb. Dose-response curves show the mean ± SEM of N = 4 test runs and were fitted using a five-parameter logistic fit. (I and J) SPR sensorgrams of C2BA5-2 and A1F3-1 binding to EBOV sGP. KD is the average ± SEM of N = 3 measurements. (K) Binding of A1F3-1 and C2BA5-2 binding to EBOV sGP, EBOV GP1, and MARV GP1. Each data point represents the average ± SEM of N = 2 replicates. Insets of (D), (I), and (J): SDS–polyacrylamide gel electrophoresis of purified scFv-Fc Abs under (1) reducing and (2) nonreducing conditions. NCP, noncontact printer; KD, equilibrium dissociation constant; MARV, Marburg virus; MW, molecular weight; kDa, kilodalton.

  • Fig. 2 Performance of the EBOV D4 assay.

    (A) (i) Photograph of a POEGMA-coated glass slide containing 24 EBOV D4 assay chips (one chip is indicated by the black box), with laser-cut acrylic and adhesive creating individual sample wells. (ii) Photograph of a single EBOV D4 assay. (iii) Fluorescence image of incubation with sample containing sGP, intense capture spots (white) and lower intensity detection spots (red) are visible. Bottom: Schematic of EBOV D4 assay. (B to F) Dose-response curves obtained using the EBOV D4 assay fabricated with A1F3-1 as cAb and C2BA5 as dAb, for EBOV sGP (B), SUDV sGP (C), BDBV sGP (D), RESTV sGP (E), and TAFV sGP (F) in FBS. Insets depict individual Ab-sGP binding profiles determined by Ag-down ELISA. (G and H) Dose-response curves using different incubation times (15 and 60 min) and sample types (pooled HS and rhesus MoS). (I) 8-log10 dose-response curve using sGP-spiked calf-serum. (J) Post-assay fluorescence stability: After running the assay, chips were incubated at 37°C and 50% humidity for 30 days. (K) Thermal stability of EBOV chips stored in pouches with desiccant at 37°C after 30 and 60 days. (L) Double-blinded spiked analyte recovery experiment in which sGP concentrations in pooled HS are determined by fluorescence intensity. In (B) to (H) and (J and K), each D4 assay data points represents the mean ± SEM of N = 4 assays; In (I), data points represent the mean of N = 3 ± SEM of N = 3 assays and in (L), data points represent the mean of N = 3 technical replicates. Ag-down ELISA data points represent the mean ± SEM of N = 2 assays. A five-parameter logistic fit (dashed line) was used to calculate LoD.

  • Fig. 3 EBOVs sGP detection in WHB.

    (A) D4 assay dose-response curves using different incubation times (15 and 60 min) for EBOV sGP spiked in WHB. (B to D) D4 assay dose-response curves for SUDV sGP (B), BDBV sGP (C), and RESTV sGP (D) spiked in WHB and incubated on the chips for 60 min. (E and F) Dose-response curves for WHB samples spiked with EBOV sGP (E) and SUDV sGP (F) and treated for 60 min at room temperature (~23°C) with Triton X-100 to a final 1% (v/v) concentration before being added to D4 chips. Treated samples were incubated on the D4 chips for 60 min. (G) Dose-response curves for blood samples spiked with EBOV sGP and treated for 30 min at 56°C with Triton X-100 to a final 1% (v/v) concentration before being added to D4 chips. Treated samples were incubated on the D4 chips for 60 min. Each data point represents the mean ± SEM of N = 4 assays; a five-parameter logistic fit (dashed line) was used to calculate LoD.

  • Fig. 4 The D4Scope, a handheld fluorescence detector.

    (A) Photograph of the D4Scope. (B) Three-dimensional design, highlighting core components and computer-on-a-chip–based architecture, with an integrated touchscreen and CMOS camera. (C) D4Scope illumination scheme, depicting oblique angle laser excitation (at a 45° angle to the surface of the D4 chip), bandpass filter, and Raspberry Pi 4 processing unit. (D) Dose-response curve obtained by plotting fluorescence intensity measured by D4Scope versus concentration of EBOV sGP spiked into pooled HS. Inset: Comparison of fluorescence readout using a GenePix scanner and the D4Scope. (E) Dose-response curve for EBOV sGP spiked into rhesus MoS imaged by a technician using the D4Scope at the Galveston National Laboratory (GNL; BSL-4) after a 30-min training session. Inset: Comparison of fluorescence readouts from SensoSpot fluorescence scanner and D4Scope at GNL. (F) Double-blinded spiked analyte recovery experiment at GNL, with sGP concentrations in rhesus MoS determined by fluorescence intensity using D4Scope and SensoSpot scanner. D4 assay data point represents the mean ± SEM of N = 4 independent assays; blinded analyte recovery data points represent the mean of N = 3 technical replicates.

  • Fig. 5 Nonhuman primate model.

    Healthy filovirus-negative rhesus macaques were challenged intramuscularly with 1000 PFU of EBOV Makona strain, n = 10. (A) Blood, plasma, and serum samples were collected at staggered time points during days 0 to 6 and tested using three assays: Serum samples were used on EBOV D4 assay, plasma samples were used for plaque assay with Vero cells, and blood samples were used in RT-PCR. The sample collection scheme and results are summarized in the panel. (B) Combined results for D4 assay, RT-PCR, and plaque assay. D4 assay: Normalized fluorescence intensity acquired with the D4Scope is reported for each time point. Each fluorescence value is the average of N = 3 technical replicates. Top: D4, PCR, and LFA results are summarized for each time point. (C) sGP concentration timeline determined using dose-response curve in Fig. 4E to convert fluorescence intensity to sGP concentration. Each data point shows the mean ± SD of sGP values for each time point. A statistically significant difference between sGP concentrations starts on day 3 (P < 0.0001; one-way ANOVA followed by Tukey’s post hoc test); values labeled with the same letter are significantly different. Inset: Western blot of serum samples from monkey 3 with sGP and GP as controls. (D) Correlation between sGP concentration and viremia determined by RT-PCR. Inset: Correlation of viral load and sGP concentration in the linear range of the EBOV D4 assay. (E) Correlation between fluorescence output measured by D4Scope and by SensoSpot scanner. Each data point is the mean ± SD of fluorescence values of each animal. RT-PCR LoD = ~103 viral genome copies/ml. Positive or negative results for PCR and D4 are color-coded. LFA results were estimated on the basis of infectious particle count results. “*” on day 4 (B) indicates one LFA with enough sensitivity to give positive results in 50% of the samples. PFU, plaque-forming units; UN, undetermined.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/13/588/eabd9696/DC1

    Materials and Methods

    Fig. S1. Schematic depiction of EBOV D4 assay.

    Fig. S2. D4 assay procedure.

    Fig. S3. Mouse IgG titers for sGP and reactivity of individual scFv-phage clones for EBOV and SUDV sGP.

    Fig. S4. scFv-Fc Ab expression, purification, and activity.

    Fig. S5. Binding constants of scFv-Fc Abs for sGP.

    Fig. S6. Ab pairing dose-response curves.

    Fig. S7. Expression and purification of A1F3-1 and C2BA5-2 monoclonal IgG2a Abs.

    Fig. S8. Binding constants of A1F3-1 (cAb) and C2BA5-2 (dAb) with multiple sGPs.

    Fig. S9. Binding profiles of other scFv-Fc Abs with sGP.

    Fig. S10. EBOV D4 assay performance.

    Fig. S11. Post-assay fluorescence stability.

    Fig. S12. EBOV D4 assay performance in human blood.

    Fig. S13. EBOV sGP lateral flow assay fabrication.

    Fig. S14. D4Scope design.

    Fig. S15. Comparison of D4Scope, GenePix, and SensoSpot performance.

    Fig. S16. RT-PCR and plaque assay.

    Fig. S17. VP30- and GP-targeting RT-PCR.

    Fig. S18. Western blot of MoS samples.

    Fig. S19. Irradiated samples from EBOV-challenged nonhuman primates.

    Table S1. D4Scope cost estimate.

    Table S2. Summary of nonhuman primate model results.

    Table S3. Summary of irradiated samples from EBOV-challenged nonhuman primates.

    Data file S1. Raw data.

    References (7580)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic depiction of EBOV D4 assay.
    • Fig. S2. D4 assay procedure.
    • Fig. S3. Mouse IgG titers for sGP and reactivity of individual scFv-phage clones for EBOV and SUDV sGP.
    • Fig. S4. scFv-Fc Ab expression, purification, and activity.
    • Fig. S5. Binding constants of scFv-Fc Abs for sGP.
    • Fig. S6. Ab pairing dose-response curves.
    • Fig. S7. Expression and purification of A1F3-1 and C2BA5-2 monoclonal IgG2a Abs.
    • Fig. S8. Binding constants of A1F3-1 (cAb) and C2BA5-2 (dAb) with multiple sGPs.
    • Fig. S9. Binding profiles of other scFv-Fc Abs with sGP.
    • Fig. S10. EBOV D4 assay performance.
    • Fig. S11. Post-assay fluorescence stability.
    • Fig. S12. EBOV D4 assay performance in human blood.
    • Fig. S13. EBOV sGP lateral flow assay fabrication.
    • Fig. S14. D4Scope design.
    • Fig. S15. Comparison of D4Scope, GenePix, and SensoSpot performance.
    • Fig. S16. RT-PCR and plaque assay.
    • Fig. S17. VP30- and GP-targeting RT-PCR.
    • Fig. S18. Western blot of MoS samples.
    • Fig. S19. Irradiated samples from EBOV-challenged nonhuman primates.
    • Table S1. D4Scope cost estimate.
    • Table S2. Summary of nonhuman primate model results.
    • Table S3. Summary of irradiated samples from EBOV-challenged nonhuman primates.
    • Legend for data file S1
    • References (7580)

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    Other Supplementary Material for this manuscript includes the following:

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