Production of monoclonal antibodies and development of an antigen capture ELISA directed against the envelope glycoprotein GP of Ebola virus

Abstract

Ebola virus (EBOV) causes severe outbreaks of Ebola hemorrhagic fever in endemic regions of Africa and is considered to be of impact for other parts of the world as an imported viral disease. To develop a new diagnostic test, monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP were characterized by ELISA, Western blot and immunofluorescence analyses. An antigen capture ELISA was established, which is specific for EBOV-Zaire and shows a sensitivity of approximately 10(3) plaque-forming units/ml. Since the ELISA is able to detect even SDS-inactivated EBOV in spiked human sera, it could complement the existing diagnostic tools in the field and in routine laboratories where high containment facilities are not available.

The Signal Peptide of the Ebolavirus Glycoprotein Influences Interaction with the Cellular Lectins DC-SIGN and DC-SIGNR

“…Samples were diluted in Laemmli sodium dodecyl sulfate (SDS) buffer, boiled at 95°C for 15 min, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes (Schleicher & Schuell, Germany), and blocked overnight at 4°C in phosphate-buffered saline containing 5% skim milk and 0.1% Tween. EBOV-GP was detected with a polyclonal rabbit serum (1:1,000), mouse monoclonal antibody 1G12 or 3B11 (34) or monoclonal anti-V5 antibody (Invitrogen, CA) at a 1:2,000 dilution, or hybridoma supernatants containing antiMyc antibody at a dilution of 1:50 and with appropriate peroxidase-coupled secondary antibodies. The expression of VP40 fusion protein was visualized with green fluorescent protein (GFP)-specific rabbit serum at a dilution of 1:5,000, and human immunodeficiency virus type 1 (HIV-1) Gag expression was detected with Gag-specific rabbit serum at a dilution of 1:1,000 and with appropriate peroxidase-coupled secondary antibodies.…”

Diagnosis of Ebola Virus Disease: Past, Present, and Future

“…ELISA antigen detection tests utilizing monoclonal antibodies against NP (22), VP40 (23), or GP (24) proteins (generated from mice immunized with purified or recombinant Ebola virus proteins) have been developed and are in place at some national reference laboratories (25), but the use of these assays for clinical diagnosis has not been reported, as realtime reverse transcription-PCR (RT-PCR) techniques have now replaced these tests (discussed in the "Real-Time RT-PCR" section below). During the recent outbreak, lateral flow immunoassays (LFIs) emerged as powerful tools for rapid antibody-mediated antigen capture that can be performed at the point of care.…”

Ebola Virus ? A Global Threat

“…VP40 plays an essential role in assembly and budding of the virus. 35 Ebola virus is susceptible to 3% acetic acid, 1% glutaraldehyde, alcohol-based products and dilutions of 5.25% sodium hypochlorite and calcium hypochlorite. The WHO recommendation for cleaning up spills of blood or body fluids is flooding the area with a 1:10 dilution of 5.25% sodium hypochlorite for 10 minutes.…”