Development, characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Journal of Virological Methods 111 (2003) 21 /28 www.elsevier.com/locate/jviromet

Development, characterization and use of monoclonal VP40antibodies for the detection of Ebola virus Andreas Lucht a,*, Roland Grunow a, Peggy Mo¨ller b, Heinz Feldmann b,c, Stephan Becker b a Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937 Munich, Germany Institute of Virology, Philipps-University Marburg, Robert-Koch-Straße 17, D-35037 Marburg, Germany c Special Pathogens Program, National Microbiology Laboratory, Health Canada, Canadian Science Centre for Human and Animal Health, Winnipeg, Canada b

Received 2 January 2003; received in revised form 3 April 2003; accepted 3 April 2003

Abstract Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the viral structural protein VP40. These mAbs were characterized by enzyme-linked immunosorbent, immunoblot and immunofluorescence assays. Subsequently, an antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Ebola virus; Viral haemorrhagic fever; Monoclonal antibodies; Diagnostics; Antigen capture ELISA

1. Introduction Ebola haemorrhagic fever is a serious emerging disease with a high fatality rate in human and nonhuman primates. After its initial recognition in Sudan and Zaire 1976 (WHO, 1976) the virus was named after the Ebola River in northern Zaire, now Democratic Republic of the Congo. The recent outbreaks of Ebola haemorrhagic fever in Uganda, Gabon and Congo have demonstrated once more the threat which Ebola virus (EBOV) poses to humans living in endemic areas of Central Africa. The introduction of EBOV Reston into the US and Italy (Jahrling et al., 1990; WHO, 1992) and the importation of Ebola haemorrhagic fever into South Africa (Okome-Nkoumou and Kombila, 1999) showed

* Corresponding author. Tel.:
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that there is certainly also a threat to public health systems in non-endemic countries. EBOV is a non-segmented, negative-stranded RNA virus and belongs to the genus Ebola-like viruses within the family Filoviridae , order Mononegavirales. Four different species of EBOV have been identified: EBOV Zaire, EBOV Sudan, EBOV Ivory Coast, and EBOV Reston (van Regenmortel et al., 2000). The inner element of a viral particle is the nucleocapsid complex which consists of the genomic RNA and four viral structural proteins: the nucleoprotein (NP), the RNAdependent RNA-polymerase (L), the polymerase cofactor VP35 and the transcription activator VP30. The matrix protein VP40 and VP24, a structural protein of unknown function, are associated with the viral envelope. The spike-like protrusions on the surface of the virions are made of homotrimers of the type 1 transmembrane glycoprotein (GP) (for review see Feldmann et al., 2001).

0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0166-0934(03)00131-9

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To date, neither a vaccine for human use nor a specific treatment for Ebola haemorrhagic fever is available. Therefore, rapid and sensitive diagnostic tools are absolutely critical for a proper and reasonable response to EBOV infections in endemic and nonendemic countries. At present, PCR and antigen detection enzyme-linked immunosorbent assays (ELISA) are the methods of choice for early EBOV diagnosis. However, rapid and reliable assays should not be restricted to a few specialized biosafety laboratories but also be available for diagnostic laboratories in the field. The main goal of this study was the development of monoclonal antibodies (mAbs) to EBOV suitable for the development of a rapid, safe and reliable antigen detecting immunoassay. Therefore, we produced mAbs directed against the viral matrix protein VP40 that makes up almost 40% of the whole viral protein mass (Elliott et al., 1985). After characterization, the mAbs were used to establish an antigen capture ELISA specific for VP40 of all four EBOV species.

2. Material and methods 2.1. Viral antigen preparation For antigen preparation, EBOV Zaire, strain Mayinga; EBOV Sudan, strain Boniface; EBOV Ivory Coast, strain Ivory Coast; EBOV Reston, strain Pennsylvania, and Marburg virus (MBGV), strain Musoke, were grown in Vero cells. The tissue culture supernatant of EBOV infected cells was cleared by centrifugation at 3000 rpm for 5 min. Then, supernatants were either used directly as antigen ( :/106 plaque forming units per ml) or concentrated (100 /500 /) and purified prior to use as published (Mu¨hlberger et al., 1999). All antigen preparations that were used in the ELISA testing were inactivated using 1% sodium dodecylsulfate (SDS). For the immunization, purified EBOV Zaire, strain Mayinga, was chemically inactivated over night with b-propiolacton (Sigma Chemicals, St. Louis, MO; final concentration 1:1000) followed by a heat inactivation step for 1 h at 60 8C in the presence of 1% Triton X-100. The concentration of viral proteins was adjusted to approximately 1 mg/ml. 2.2. Development of monoclonal antibodies Six-week-old female BALB/c mice (Charles River, Sulzfeld, Germany) were immunized intraperitoneally four times with inactivated EBOV Zaire antigen at day 0, 25, 48 and 53 using Titermax (Serva, Germany) as an adjuvants according to the local animal use regulations. The immune response to the structural proteins of

EBOV was assessed by immunoblot analysis. Three animals with the highest titres were splenectomized at day 3 after the last antigen injection. The hybridoma technique was used for the development of specific mAbs (Ko¨hler and Milstein, 1975; Grunow et al., 1990). The spleen cells were fused with the X63Ag8.653 myeloma cell line (Kearney et al., 1979) at a proportion of 10:1 using polyethylene glycol 1500 (Life Technologies, Pairley, Scotland). The hybridoma cells were cultured in DMEM/RPMI 1640 medium (ratio 1:1) supplemented with 2.85 g/l NaHCO3, 1 mM NaPyruvat, MEM nonessential amino acids solution, 2 mM L-glutamine (all from Life Technologies), 10% fetal calf serum, 2% ESG hybridoma growth factor (both from Costar Biologicals, The Netherlands), and Antibiotic/Antimycotic (Life Technologies). Fusion products were seeded in 96-well plates (Nunc, Wiesbaden, Germany). Hybridoma cells were selected by hypoxanthine, aminopterin, and thymidine (HAT, Life Technologies) during the first 3 weeks, and hypoxanthine, and thymidine medium (HT, Sigma Chemicals) for further 3 weeks. For expansion, cells were cultivated in 24- and in 6-well plates (Nunc) and finally in small tissue culture flasks (Nunc) at 37 8C and 7.5% CO2. Hybridoma cell lines producing specific antibodies were recloned four to six times by limiting dilution to ensure monoclonality.

2.3. Screening ELISA Antibody production in hybridoma cells was detected using an ELISA technique. Briefly, purified EBOV Zaire antigen (inactivated by 1% SDS) was diluted 1:1000 in carbonate buffer (pH 9.0) and coated to the solid phase of 96-well plates (Nunc-Immuno MaxiSorpTM plates, Nunc) at 4 8C overnight. As negative control, tissue culture supernatant of non-infected Vero cells was used. To avoid unspecific binding the plates were incubated with 5% skim milk/0.05% Tween 20 in aqua dest at 37 8C for 60 min. Fifty microlitre hybridoma supernatant were used undiluted, or diluted in PBS 1:10, and incubated at 37 8C for 60 min. Bound antibodies were detected using POD-conjugated goat anti-mouse immunoglobulin (DAKO, Hamburg, Germany; diluted 1:20 000 in PBS) for 60 min at 37 8C followed by tetramethyl benzidine (TMB) substrate solution (Seramun, Dolgenbrodt, Germany) for 15 min at room temperature. The reaction was stopped with 0.25 M H2SO4. The optical density (OD) was measured at 450 nm using a Titertek Multiscan PlusTM plate reader (Labsystems, Benediktbeuren, Germany). After each incubation step, plates were washed five times with PBS, containing 0.05% Tween 20.

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2.4. Characterization of monoclonal antibodies

2.7. Antigen capture ELISA

The specificities of the mAbs were evaluated by immunoblot analysis using antigen from all known EBOV species and MBGV (for preparation see above) as described elsewhere (Becker et al., 1992, 1996). Briefly, viral antigen preparations were separated on 10% SDS-PAGE and blotted onto nitrocellulose. The nitrocellulose was incubated with hybridoma cell culture supernatant for 2 h at room temperature and reactivity was detected using a POD-conjugated goat anti-mouse antibody (DAKO; dilution 1:1000). In order to define better the specificity of the mAbs, HeLa cells were infected with the recombinant vaccinia virus MVA-T7 and subsequently transfected with plasmids containing the genes for the EBOV structural proteins GP, NP, VP40, VP35, and VP30 (Becker et al., 1996). At 12 h after transfection, the cells were lysed and prepared for immunoblot analysis as described above. For confirmation purposes, we investigated the reactivity of the mAbs by indirect immunofluorescence. HeLa cells, grown on glass cover slides, were infected and transfected as described above. Cells were fixed with 3% paraformaldehyde at 8 h p.i. and subjected to immunofluorescence using mAbs and a rhodamine-conjugated anti-mouse antibody as published previously (Becker et al., 1998).

The mAbs detecting VP40 of all EBOV species were used to develop an antigen capture ELISA. Briefly, 96well plates (Nunc-Immuno MaxiSorpTM plates, Nunc) were coated overnight at 4 8C with one of the appropriate mAbs (5F6, 2C4, 5A5, 3F2, 2G2 or 3G7) in carbonate buffer (pH 9.0). After five washes with PBS/ 0.05% Tween 20, plates were incubated with PBS containing 5 or 10% of various supplements with or without 0.05% Tween 20 at 37 8C for 60 min to prevent nonspecific binding. Following washing, EBOV antigen (diluted in PBS 1:1000) or as indicated, was added and incubated for 1 h at 37 8C. Subsequently, plates were washed and incubated at 37 8C for 60 min with the biotin-labelled mAb 5F6 (diluted in PBS/1% skim milk/ 0.05% Tween 20). Thereafter, plates were washed and incubated for 60 min at 37 8C with Streptavidin-POD conjugate (Amersham International, Amersham, UK; diluted 1:4000 in PBS/1% skim milk/0.05% Tween) followed by TMB substrate solution for 15 min at room temperature. The reaction was stopped by 50 ml 0.25 M H2SO4. OD was measured at 450 nm with a reference wavelength of 620 nm using a DigiScanTM plate reader (Asys Hitech, Eugendorf, Austria) and MIKROTM WIN 2000 software (Mikrotek, Overath, Germany). For both, antigen in cell culture medium and TM serum, cut-off levels were set by calculating the mean of ten negative controls plus three standard deviations.

2.5. Quantification and subclass determination of immunoglobulins The amount of mAbs in cell culture supernatant was quantified using the anti-mouse Ig hybridoma screening reagent (Boehringer, Mannheim, Germany) and a mouse IgG standard (Sigma Chemicals). Immunoglobulin subclasses were determined using the ‘Isostrip Mouse Monoclonal Antibody Isotyping Kit’ (Boehringer) and the ‘ImmunoPure Monoclonal Antibody Isotyping Kit’ (Pierce, Rockford, IL). 2.6. Large-scale production and purification of specific mAbs The cell lines producing the VP40-specific mAbs 2C4 and 5F6 were adapted to protein-free PFHM II Medium supplemented with Glutamax 2 (Life Technologies). Cells were first grown in small tissue culture flasks (Nunc) and then expanded in the Tecnomouse production system (Integra Biosciences, Lowell, MA) using protein-free medium supplemented with high glucose (4.5 g/l), high glutamine (4 mM) and 150 mg/l gentamycin (all Life Technologies). For purification the mAb Trap GII Kit (Amersham Pharmacia, Freiburg, Germany) was used. The mAb 5F6 (2 mg) was labelled with biotin using a Biotin Labelling Kit (Boehringer). Small amounts of the mAbs 5A5, 3F2, 2G2, 3G7 were produced and purified as well.

3. Results 3.1. Production and characterization of mAbs Balb/c mice were immunized with inactivated antigen of EBOV Zaire. Following three booster immunizations, mouse sera were titrated against EBOV Zaire antigen using immunoblotting. The three mice with the highest antibody titres were splenectomized and the spleen cells were fused with a myeloma cell line. After repeated cloning, hybridoma cell lines were selected producing specific mAbs to structural proteins of EBOV as determined by ELISA. Each hybridoma cell line produced 10 /100 mg immunoglobulins per ml cell culture supernatant. The specificity of the mAbs was investigated by immunoblot and indirect immunofluorescence. For further analysis, 10 mAbs were selected reacting with EBOV-specific proteins in the range of 30/40 kDa. This approach did not determine the definite protein specificity. Therefore, we tested the mAbs by immunoblot against specific viral antigens derived from transfected HeLa cells. Nine mAbs, 5F6, 2C4, 3F2, 5A5, 2G2, 3G7, 1F3, 4D7 and 3F8, reacted with the matrix protein VP40 (Fig. 1A and B, lanes 1 and 2). Using the same antibody

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plasmids encoding the different EBOV proteins. The mAbs, recognizing VP40 or VP30 in immunoblot, showed a rather homogeneous or reticular pattern of staining (Fig. 2) as seen with cytoplasmic proteins. These localizations are expected for the distribution of the respective recombinant proteins (Martin-Serrano et al., 2001; Modrof et al., 2002). The obtained nine mAbs directed against VP40 were of different immunoglobulin subclasses: six of IgG1, two of IgG2a, one of IgG2b. The mAb against VP30 was of IgM subclass (Table 1, column 2). All of them possessed kappa light chains. To characterize further the mAbs we examined the cross-reactivity to other EBOV species and to MBGV using immunoblot analysis and ELISA. Six out of nine VP40-specific mAbs (5F6, 2C4, 5A5, 2G2, 3F2, 3G7) recognized all four known EBOV species. VP40-specific mAb 1F3 detected EBOV Zaire and Sudan but not Ivory Coast or Reston. The remaining two VP40specific mAbs (3F8, 4D7) were monospecific to EBOV Zaire VP40. The IgM mAb 4C7 recognized VP30 of all known EBOV species. None of the mAbs cross-reacted with MBGV VP40. The ELISA results confirmed the data obtained by immunoblot (Table 1).

Fig. 1. Reactivities of mAbs in immunoblot analysis using virionassociated EBOV Zaire proteins and recombinant VP40 and VP30 derived from EBOV Zaire. HeLa cells were infected with EBOV or the recombinant vaccinia-virus MVA-T7 and transfected with plasmids containing the genes of VP40 or VP 30. At 12 h p.i., cells were lysed; lysates were separated by SDS-PAGE and electroblotted onto PVDF membranes. Membranes were incubated with the respective mAbs. Lane 1: EBOV virion-associated proteins; lane 2: EBOV recombinant antigens; lane 3: wild-type vaccinia virus proteins; lane 4: mockinfected cells. (A) VP40-specific mAb 5F6; (B) VP40-specific mAb 2C4; (C) VP30-specific mAb 4C7.

3.2. Development of the antigen capture ELISA Based on the specific reactivity of the different mAbs, a cross-reactive antigen capture ELISA detecting all known EBOV species was developed. Hybridoma cells secreting mAbs that reacted strongly with VP40 of all EBOV species were selected (5F6 and 2C4), and adapted to protein-free medium. Using the Tecnomouse production system it was possible to produce 2.5 mg/ml 5F6 and 4 mg/ml 2C4. Both mAbs could be purified and labelled with biotin without any considerable loss of activity (data not shown). At first, the most suitable mAbs for the ELISA had to be identified. Using the same mAb 5F6 for coating and

concentration, four clones (2C4, 5F6, 3F2, 3F8) reacted strongly with the authentic and the recombinant VP40, whereas the remaining five mAbs (1F3, 5A5, 2G2, 3G7 and 4D7) showed rather weak signals. The mAb 4C7 (IgM) recognized the viral transcription factor VP30 (Fig. 1C, lanes 1 and 2, (Mu¨hlberger et al., 1999)). The reactivity of the different mAbs is summarized in Table 1. The protein-specific reactivity of the mAbs was confirmed by indirect immunofluorescence on HeLa cells infected with MVA-T7 and transfected with Table 1 Characterization of the mAbs against viral structural proteins of EBOV mAb

5F6 2C4 3F2 5A5 2G2 3G7 1F3 4D7 3F8 4C7

Immunoglobulin subclass

IgG2a IgG1 IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a IgM

Recognized viral protein

VP40 VP40 VP40 VP40 VP40 VP40 VP40 VP40 VP40 VP30

Cross-reactivities EBOV Zaire

EBOV Sudan

EBOV Ivory Coast

EBOV Reston


/
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/
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/ / / /
/

Immunoglobulin subclasses of the mAbs, recognized viral structural proteins and patterns of cross-reactivities with all known EBOV species in ELISA and immunoblot analysis. Reactivity was recorded as absent (/) or present (
/).

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Fig. 2. Immunofluorescence analysis of mAbs. HeLa cells were infected with MVA-T7 and transfected with plasmids encoding EBOV structural proteins VP40 or VP30. Infected cells were fixed with 3% paraformaldehyde at 8 h p.i. Cells were incubated with mAbs directed against the respective proteins and with a rhodamine-conjugated donkey anti-mouse antibody. Mock-infected cells and wild-type vaccinia virus infected cells were used as negative control. (A) VP40-specific mAb 5F6; (B) VP40-specific mAb 2C4; (C) VP30-specific mAb 4C7.

detection did not work. Other combinations of mAbs showed better results, and the best combination was mAb 2C4 for coating and mAb 5F6 for detection. This result suggested that these mAbs recognize different epitopes on VP40. Then concentrations of antibodies were determined that showed the optimal signal to noise ratio in the antigen capture ELISA. The best results were found when 10 mg/ml 2C4 were coated as the capture antibody and 2 mg/ml biotin-labelled 5F6 were used for detection. For blocking, 10% skim milk in PBS/ 0.05% Tween was deemed most suitable. The cut-off was determined and set to an OD of 0.15. The detection limit of the ELISA was determined as 1:1 024 000 for purified EBOV particles and 1:8000 for antigen derived from supernatants of infected tissue cultures, which corresponds to approximately 102 p.f.u./ml (open circles, Fig. 3A and B). The ELISA was genus-specific and detected all known EBOV species. In the following, the ELISA was evaluated using sera spiked with EBOV. Supernatant from EBOV infected Vero cell culture was added to human sera and tested in different dilutions of the supernatant in serum. The cutoff for serum samples was set to an OD of 0.2. The sensitivity of the assay was a final dilution of the tissue culture supernatant of 1:500 in serum (circles, Fig. 4). To check whether SDS-inactivation impaired the sensi-

tivity of the ELISA, 1% SDS was added to the spiked serum samples that were then tested. The measured ODs decreased but the sensitivity of the assay fell only slightly (1:250) (open squares, Fig. 4). The sensitivity of the assay corresponded to 103 /104 p.f.u./ml serum.

4. Discussion Since the first outbreaks of Ebola haemorrhagic fever 1976 in Zaire and Sudan several efforts have been made to develop more reliable diagnostic tools. In spite of many improvements in recent years, all antibody detection assays, including the sensitive and specific IgM capture ELISA (Ksiazek et al., 1999b) face the problem that early laboratory diagnosis is impaired by the relatively late or weak humoral immune response to EBOV infection (Rowe et al., 1999). Although IgM can occasionally be detected as early as 2 days post onset of symptoms, it usually does not occur before the end of the first week and, thus, correlates with deaths in fatal cases. IgG levels do not rise before day 6/18 and are therefore not suitable markers for acute diagnosis. Because of the presence of high titres of infectious virus in blood and tissues, antigen detection plays the most important role in early detection of EBOV

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Fig. 3. ELISA results with purified antigen and tissue culture supernatant of infected Vero cells. (Measured ODs /3 were shown as an OD of 3 in this figure.) (A) Purified antigen. (k) EBOV Zaire; (') MBGV; (I) Mock. (B) Tissue culture supernatant of infected Vero cells. (k) EBOV Zaire; (') MBGV; (I) Mock.

infection. Viral antigen is detectable as early day 3 after the onset of symptoms (Rowe et al., 1999), and at this stage of the infection antigen titres are similar in fatalities and survivors. During the following days, antigen titres decrease in survivors and finally disappear with recovery, whereas in fatalities antigen load increases until death (Baize et al., 1999). Several assays

Fig. 4. Detection of antigen in human serum spiked with EBOV Zaire. (Measured ODs /3 were shown as an OD of 3 in this figure). (m) Untreated serum; (I) 1% SDS-inactivated serum.

have been developed for antigen detection which all have limitations in their application. Among these are virus isolation, which is time-consuming and needs biocontainment (Bowen et al., 1977; Pattyn et al., 1977), electron microscopy, which is less sensitive and needs special equipment (Geisbert and Jahrling, 1995), direct immunofluorescence stainings of blood and impression smears from organs, which is rapid and sensitive but sometimes difficult to interpret (Rollin et al., 1990), and immunohistochemistry, which is especially used for post mortem diagnosis (Zaki et al., 1999). At present, the most favourable methods for early diagnosis of Ebola haemorrhagic fever are RT-PCR and antigen detection ELISA. RT-PCR is a rapid and sensitive method for the detection of viral genomic RNA, which is able to detect virus from early acute disease throughout early recovery (Leroy et al., 2000; Sanchez and Feldmann, 1996; Sanchez et al., 1999). However, verification of positive results is essential due to the potential risk of contamination (Lenz et al., 1998). Fast and sensitive antigen detecting ELISAs are alternative and confirmatory assays for diagnosis. Most of these antigen capture ELISAs are based on the use of a combination of monoclonal capture and polyclonal detection antibodies (Borisevich et al., 1996; Ksiazek et al., 1992, 1999a; Merzlikin et al., 1995; Niikura et al., 2001). Polyclonal antibodies often show higher sensitivity and avidity, but mAbs have the advantages of higher specificity and unlimited availability. Therefore, we developed an antigen capture ELISA using monoclonal capture and detection antibodies directed against VP40 of EBOV. The matrix protein VP40 seemed to be a promising target for the following reason: VP40 is the major viral protein. It has been estimated that one virion contains approximately 3600 VP40 molecules what is 37.7% of its whole protein mass (Elliott et al., 1985). The antigen capture ELISA used only specific mAbs for capture as well as for detection of EBOV antigen. Two mAbs, recognizing VP40 of all EBOV species, were used for the development of an EBOV genus-specific test system. Initial investigations showed that the mAb 2C4 was most suitable as the capture antibody, and the biotin-labelled mAb 5F6 for detection. The supernatant of infected tissue culture could be detected up to 1:8000, which corresponds to approximately 102 p.f.u./ml. The purified EBOV Zaire antigen could be diluted approximately up to 1:1 024 000. During the purification procedure of viral antigen, the amount of virions is approximately 100/500/ concentrated compared to tissue culture supernatant. That fits well with the two different detection limits of tissue culture supernatant and purified antigen (ratio 1:128). EBOV antigen could be detected in spiked human sera. The ELISA has a sensitivity of approximately 103 / 104 p.f.u./ml serum. This seems to be somewhat lower than approximately 102 p.f.u./ml, which has been found

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for an antigen capture ELISA described before (Ksiazek et al., 1992). The inactivation of sera with 1% SDS did not lead to a significant loss in sensitivity in our ELISA. In respect to biosafety, this is an improvement over current diagnostic protocols and very promising for the use in the field. Assuming that a patient suffering from Ebola haemorrhagic fever has serum titres of approximately 106 /108 viral particles per ml, it is reasonable to expect that the sensitivity of the ELISA is sufficiently high to detect even early stages of infection. In conclusion, the mAbs to EBOV were suitable for developing a specific and sensitive antigen detection system. Most promising is the fact that even SDSinactivated material could be detected with this antigen capture ELISA. Inactivation of the samples with SDS could be readily accomplished under field conditions, thereby reducing remarkably the risk to be faced by the investigators. Therefore, this test might be a valuable tool for rapid detection of EBOV and Ebola haemorrhagic fever diagnosis in the field and in the laboratory.

Acknowledgements The work was supported by the Bundesministerium der Verteidigung (Sonderforschungsauftrag 23Z1-S439902), by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 286, TP A6 and Sonderforschungsbereich 535 TP A4 and B9) and the Canadian Institutes of Health Research (MOP-43921). Thanks to C. Otterbein, N. Romhart and E. Zeman.

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