With the upsurge in international traffic, the chance of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all around the globe. subtypes. These total results claim that our ELISA system should use three of 4 Ebola subtypes. Furthermore, our ELISA program recognized the NP in subtype Reston-infected monkey specimens, as the history level in non-infected specimens was suprisingly low, A 922500 recommending the usefulness from the ELISA for lab diagnosis with medical specimens. Ebola disease infection causes one of the most severe hemorrhagic fevers and has a high fatality rate (20). Although Aviptadil Acetate 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, C?te 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 A 922500 antibody production is observed at the onset of clinical symptoms (1, A 922500 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 way to obtain that ELISA system is bound rather. For these good reasons, we made a decision to establish another operational program for the recognition of Ebola viral antigen. Toward A 922500 this objective, we first founded MAbs to a recombinant nucleoprotein (rNP) of Ebola pathogen subtype Zaire. NP is among the main viral structural parts and includes 739 amino acidity (aa) residues. It really is expected how the hydrophobic N terminus of the proteins may be involved with genomic RNA binding, as the hydrophilic and intensely acidic C terminus could be mixed A 922500 up in binding of additional viral protein, analogous to paramyxovirus (13, 17). We chose this molecule for the target of antigen detection because of the abundance of NP in Ebola virus particles and the availability of cDNA and sequence information. Here, we report on the successful development of an antigen-capture sandwich ELISA system with a novel NP-specific MAb which recognizes 26 aa residues on the C terminus of NP. MATERIALS AND METHODS Cell culture. Hybridomas and their parental cell line, P3/Ag568, were maintained in RPMI 1640 (Gibco BRL, Rockville, Md.) supplemented with 10% fetal bovine serum, nonessential amino acids (Gibco BRL), and antibiotics (streptomycin and penicillin; Gibco BRL). Hypoxanthine-aminopterin-thymidine supplement (Gibco BRL) was added to the medium during the selection of hybridomas, as recommended by the supplier. Tn5 insect cells were maintained in TC100 (Gibco BRL) supplemented with 10% fetal bovine serum, 2% tryptose phosphate broth (Difco, Detroit, Mich.), and kanamycin. Clinical specimens. Tissues and sera from cynomolgus monkeys (with the pGEX2T vector (Amersham Pharmacia, Little Chalfont, United Kingdom) after PCR amplification (18). The primers used in the study are summarized in Table ?Table1.1. To express the 26-aa peptides of the Sudan and Reston subtypes, primers SNP8EF.