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. 2015 Oct 1;212 Suppl 2(Suppl 2):S359-67.
doi: 10.1093/infdis/jiv353. Epub 2015 Jul 30.

Development of Prototype Filovirus Recombinant Antigen Immunoassays

Affiliations

Development of Prototype Filovirus Recombinant Antigen Immunoassays

Matt L Boisen et al. J Infect Dis. .

Abstract

Background: Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples.

Methods: Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development.

Results: Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone.

Conclusions: The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.

Keywords: ELISA; Ebola; Ebola virus; filovirus; lateral flow immunodiagnostic; point-of-care testing.

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Figures

Figure 1.
Figure 1.
Recombinant filovirus protein expression in mammalian and bacterial cells. From left, Ebola virus (EBOV) glycoprotein (GP; mucin-containing), EBOV GP (mucin-deleted), EBOV secreted GP (sGP), Sudan virus (SUDV) GP (mucin-deleted), SUDV sGP, EBOV nucleoprotein (NP), Marburg virus (MARV) NP, EBOV VP40, and MARV VP40. GPs were expressed in either human 293T cells or in recombinant insect cell systems; those shown here were purified from 293T in multimilligram quantities for crystallization. NP and VP40 antigens were produced in Escherichia coli.
Figure 2.
Figure 2.
Escherichia coli–produced recombinant VP40 and Ebola virus (EBOV) virus-like particles (VLPs) precipitated from supernatants of HEK-293T/17 cell cultures transfected with EBOV VP40, EBOV nucleoprotein (NP), and EBOV glycoprotein (GP) mammalian expression constructs were resolved on nonreducing Bis-Tris/MES sodium dodecyl sulfate polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and probed with goat anti-VP40 or goat anti-NP polyclonal antibodies (VHFC) and a rabbit anti-goat immunoglobulin G (H + L)-HRP (KPL). Blots were imaged with LumiGlo chemiluminescent substrate (KPL) in a GE ImageQuant LAS4000 imager.
Figure 3.
Figure 3.
Detection of Ebola virus (EBOV) VP40–specific and Sudan virus (SUDV) VP40–specific immunoglobulin G (IgG) from rhesus macaque serum. Microplates were coated with either EBOV or SUDV VP40 and then overlaid with serial dilutions of serum from a rhesus macaque infected with EBOV. IgG contained in the serum was captured over a dilution range of approximately 2 logs, using either EBOV (black) or SUDV (red) VP40. No immunoglobulin M (IgM) was captured by EBOV (blue) or SUDV (green) over the same range because the IgM response had subsided at the time of serum collection.
Figure 4.
Figure 4.
Detection of Ebola virus (EBOV)–specific immunoglobulin G (IgG) from patients with suspected Lassa fever presenting to the Kenema Government Hospital Lassa fever ward between 9 April 9 2014 (G3642) and 4 June 2014 (G3718). Microplates were coated with either EBOV VP40 or glycoprotein (GP) or with a combination of both antigens and then were overlaid with serial dilutions of serum from patients with suspected Lassa fever. Polyclonal anti-human IgG–horseradish peroxidase was used as a detection reagent. Blood specimens obtained sequentially from most patients during hospitalization permitted analysis of seroconversion and increasing IgG titers to EBOV antigens. A single blood specimen was available from patient G3642. Sera were analyzed over a titration range, as permitted by available sample volumes. Patient G3670, who survived Ebola, showed a clear rise in titer between the first and second blood specimen collected, although precise end point titers could not be determined because the samples were too small for analysis. Patients G3710 and G3718, who also survived EBOV infection, registered a moderate increase in IgG titers to both EBOV antigens. Patient G3717, who died from Ebola, registered a substantial titer in the first blood specimen, which did not increase significantly over time. Patient G3713, who also died, did not register a significant titer to EBOV antigens throughout the interval during which samples were collected. IgG reactivity to VP40 (blue), GP (red), and the combination of both antigens (green) from duplicate sample analysis is plotted with standard deviations.
Figure 5.
Figure 5.
Prototype Ebola virus (EBOV) and Sudan virus (SUDV) antigen-capture enzyme-linked immunosorbent assay (ELISAs) detect EBOV and SUDV VP40. Microplates coated with capture antibodies were challenged with serial dilutions of either EBOV VP40 or SUDV VP40. A, Detection of EBOV VP40 with pair 1 (black), pair 2 (blue), or pair 3 (red) had nearly identical curves; EBOV VP40 detected with pair 4 (green) had a slightly reduced sensitivity. B, Detection of SUDV VP40 with pair 1 (black) showed greater variability, detection with pair 2 (blue) and pair 4 (green) had similar sensitivity, and detection with pair 3 (red) had the lowest sensitivity. C, Prototype Marburg virus (MARV) antigen-capture ELISA detected MARV VP40. Microplates coated with goat-α-MARV antibodies were challenged with serial dilutions of MARV VP40. Detection of captured MARV VP40 with the same self-pairing goat-α-MARV antibody (pair M1; black) showed dose-dependent detection, which lacked an upper plateau over the chosen dilution range.
Figure 6.
Figure 6.
Prototype Ebola virus (EBOV)/Sudan virus (SUDV) antigen-capture lateral flow immunodiagnostic (LFI) assays detect EBOV and SUDV VP40. LFI strips prepared with pair ES5 were challenged with a serial dilution (300-0.1 µg/mL) of either EBOV VP40 antigen (A) or SUDV VP40 antigen (B). Pair ES5 detected both antigens in a dose-dependent manner, with greater qualitative sensitivity observed for EBOV VP40 antigen. C, Prototype Marburg virus (MARV) antigen-capture LFI assay detects MARV VP40. LFI strips prepared with pair M2 were challenged with a serial dilution of MARV VP40 antigen. Pair M2 detected the antigen in a dose-dependent manner. Control (C) and test (T) lines on each set of strips are marked. Abbreviation: BCN, bulk control normal.

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