Detection of Ebola Viral Antigen by Enzyme-Linked Immunosorbent Assay Using a Novel Monoclonal Antibody to Nucleoprotein

Abstract

With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.Ebola virus infection causes one of the most severe hemorrhagic fevers and has a high fatality rate (20). Although the region of endemicity of Ebola virus is limited, the risk of infection of humans and animals in other parts of the world is increasing with the increase in international traffic and transactions. Since Ebola virus causes secondary human-to-human infections among medical personnel and family members (2,20), it is important to diagnose the infection at the early stage of an outbreak and to alert society.On the basis of genetic divergence, four subtypes of Ebola viruses have been defined: subtypes Zaire, Sudan, Côte d 'Ivoire, and Reston (3,5,14). The first three subtypes cause severe clinical symptoms in both humans and monkeys, while subtype Reston has caused disease only in monkeys (4, 10, 11). Ebola virus infection has an acute onset, and frequently, no antibody production is observed at the onset of clinical symptoms (1, 7). On the other hand, the virus load in patients' blood and tissues such as liver is extremely high (7). Therefore, quick and accurate primary screening for Ebola virus infection can be achieved by detection of the viral antigens rather than by detection of specific antibodies (14).An antigen-detection system for Ebola virus infection was reported and successfully applied in the field (6). However, the information on that enzyme-linked immunosorbent assay (ELISA) is quite limited. For example, the monoclonal antibodies (MAbs) used in that system have not been reported even in terms of their molecular specificities. Moreover, the supply of that ELISA system is rather limited. For these reasons, we decided to establish another system for the detection of Ebola viral antigen. Toward this goal, we first established MAbs to a recombinant nucleoprotein (rNP) of Ebola virus subtype Zaire.NP is one of the major viral structural componen...

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.…”

Antigen Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Reston Ebola Virus Nucleoprotein

“…MAbs were prepared as described previously (29). Briefly, BALB/c mice were subcutaneously immunized with His-EBO-R-NP together with Freund complete adjuvant (Becton Dickinson, Franklin Lakes, N.J.) and boosted subcutaneously three times with IMJECT-ALUM (Pierce, Rockford, Ill.) at 2-week intervals.…”

“…Antigen capture ELISA was performed as described previously (29). Briefly, a microtiter plate (Becton Dickinson) was coated with purified MAbs (100 ng per well in 100 l of PBS) overnight at 4°C.…”

“…4030-035; Gibco BRL) according to the manufacturer's instructions. The supernatants of the hybridoma cells were screened in an immunoglobulin G (IgG) ELISA (29) with His-EBO-R-NP as an antigen. Crude hybridomas were isolated from ELISA-positive wells, and the hybridoma clones were established by the limiting dilution method.…”

Ebola Virus: A Scientometric Study of World Research Publications

“…[10] Nevertheless, the antigen-detection tests with filovirus-specific mAb could be one of the best ways for diagnosis of filovirus infections. [18,19] The Objectives of the Study…”