Jpn. J. Infect. Dis., 59, 323-325, 2006

Short Communication Nucleoprotein-Capture Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies to Recombinant Nucleoprotein: Detection of Authentic Marburgvirus Masayuki Saijo*, Marie-Claude Georges-Courbot1, Shuetsu Fukushi, Tetsuya Mizutani, Marianneau Philippe1, Alain-Jean Georges2, Ichiro Kurane and Shigeru Morikawa Department of Virology 1, National Institute of Infectious Diseases, Tokyo 208-0011, Japan; 1Unit of Biology of Viral Emerging Infections, Institute Pasteur; and 2Laboratory P4 Jean-Merieux-Inserm, Lyon, France (Received April 26, 2006. Accepted June 19, 2006) SUMMARY: There have recently been large outbreaks of hemorrhagic fever (MHF) caused by Marburgvirus (MARV) in the Democratic Republic of Congo and Angola. The development of reliable diagnostic systems for MHF is urgently needed. An antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) using either of the two monoclonal antibodies (2A7 and 2H6) produced by immunizing mice with recombinant nucleoprotein of MARV was described (Journal of Medical Virology, 76, 111-118, 2005). In the present study, it was revealed that the Ag-capture ELISA specifically detected authentic MARV antigen and that the sensitivity of the Ag-capture ELISA was at a level similar to that of reverse-transcription polymerase chain reaction. These results suggest that the Ag-capture ELISA using the monoclonal antibodies, 2A7 and 2H6, is applicable to the diagnosis of MHF.

Lake Victoria Marburgvirus (MARV) infections cause [PFU]/ml determined by a plaque-forming assay in Vero cells) Marburg hemorrhagic fever (MHF), one of the most severe was used. All procedures requiring the manipulation of forms of hemorrhagic fever, with a high mortality rate (1-3). infectious MARV were carried out in a maximum safety labo- MARV belongs to the Marburgvirus, family . ratory (Biosafety Level-4 laboratory) in INSERM (P4 Jean The first documented outbreak of MHF occurred in the former Merieux Inserm) in Lyon, France. Two monoclonal anti- West Germany and the former Yugoslavia in 1967 (4). After bodies (2A7 and 2H6), which reacted with the carboxy- the first documented outbreak, 3 sporadic cases of MHF were terminal part of the MARV-rNP, were used (8). The protein reported in Zimbabwe (1975) and Kenya (1980 and 1987) concentration of the purified monoclonal antibody solu- (3,5-7). From 1998 to 1999, there was a large outbreak in the tions for 2A7 and 2H6 was determined to be 570 μ/ml and Democratic Republic of Congo, formerly Zaire (1). The 470 μ/ml, respectively. The MARV solution was diluted with largest outbreak of MHF to date started in 2004, in which serum before testing and mixed with the same more than 300 patients have been reported and with a volume of phosphate-buffered saline (PBS) containing 2% mortality rate of over 90% in Uige Province in Angola (see triton-X, and then subjected to Ag-capture ELISA. The WHO website: http://www.who.int/csr/don/2005_05_18a/en/ Ag-capture ELISA using either of the monoclonal antibodies, index.html). Therefore, the development of diagnostics for 2A7 and 2H6, was carried out as reported previously (8). MHF is urgently needed. Purified monoclonal antibody was coated on microwell In our previous report, we characterized two monoclonal immunoplates (Falcon; Becton Dickinson Labware, Franklin antibodies to the recombinant nucleoprotein (rNP) of MARV, Lakes, N.J., USA) at the designated concentration of mono- 2A7 and 2H6 (8). These antibodies were useful in the antigen- clonal antibody in PBS (100 ng/100 μl/well) at 4°C over- capture enzyme-linked immunosorbent assay (Ag-capture night, followed by blocking with PBS containing 5% nonfat ELISA). However, the efficacy of the Ag-capture ELISA milk and 0.05% Tween-20 (PBST-M) for 1 h at room tempera- using the authentic antigen was not evaluated. In the ture (RT). After the plates were washed with PBS containing present study, to confirm the usefulness of the Ag-capture 0.05% Tween-20 (PBST), 100 μl of serially diluted samples ELISA for the diagnosis of MHF, we examined the assay with was added and the plates were incubated for 1 h at 37°C. The each of the monoclonal antibodies to detect the authentic plates were then washed with PBST, and 100 μl of rabbit virus antigen (MARV Musoke strain). We also compared the polyclonal antibody raised against rNP of MARV diluted Ag-capture ELISA with reverse-transcription polymerase 1:500 with PBST-M was added to each well (9). After 1 h chain reaction (RT-PCR) in terms of sensitivity for the detec- incubation at 37°C, the plates were washed with PBST and tion of MARV. horseradish peroxidase-conjugated goat anti-rabbit IgG MARV, Musoke strain (2.5 × 107 plaque-forming units (Zymed Laboratories Inc., South San Francisco, Calif., USA) was added. The plates were incubated for 1 h at RT. After *Corresponding author: Mailing address: Special Pathogens Labo- another extensive wash with PBST, 100 μl of ABTS sub- ratory, Department of Virology 1, National Institute of Infec- strate solution (4 mM 2,2’-azino-di-[3-ethylbenzthiazolin tious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208- sulfonate (6)] (Roche Diagnostics, Mannheim, Germany) 0011, Japan. Tel: +81-42-561-0771 ext. 320, Fax: +81-42-561- was added and the optical density (OD) was measured at a 2039, E-mail: [email protected] wavelength of 405 nm with a reference wavelength of 490

323 nm after 30 min of incubation at 37°C. As a negative control, 94°C for 5 min, 3 cycles of 94°C for 30 s, 37°C for 30 s and mock antigen-inoculated wells were also tested. The OD of 72°C for 30 s, followed by 72°C for 2 min, 30 cycles of 94°C the MARV sample showed high values in the Ag-capture for 30 s, 45°C for 30 s, and 72°C for 30 s, followed by 72°C ELISA with either of the monoclonal antibodies, 2A7 and for 5 min. Ten microliters of the reaction mixture was sub- 2H6, at dilutions from 1:8 to 1:213 and from 1:8 to 1:211, jected to 2% agarose gel-electrophoresis, and the amplified respectively, in a dilution level-dependent manner, while the DNA was visualized by the standard method using ethidium samples showed very low OD values in Ag-capture ELISA bromide. Although the data are not shown here, the RT-PCR without the capture monoclonal antibodies (Fig. 1A and 1B). using this primer set was the most sensitive among the RT- Negative control samples, human sera without MARV, also PCRs using either of the 3 primer sets, Filo-A/Filo-B, mbg1 showed very low OD values in the Ag-capture ELISA with (5´-ACTCTCCAGAAGACAGAAGA-3´)/mbg2 (5´-AGCG the monoclonal antibodies 2A7 and 2H6 (Fig. 1A and 1B). ATGGGCTTTCAGGACA-3´), or mbg3 (5´-AGCGATGGG The newly developed Ag-capture ELISA could detect the CTTTCAGGACA-3´)/mbg4 (5´-CGGTACATTGTTGTGGA authentic MARV nucleoprotein at a level equivalent to the GGC-3´). The MARV sample demonstrated positive reactions concentration of MARV nucleoprotein from approximately at dilutions from 1:101 to 1:104 in RT-PCR assay (Fig. 1C). 3 × 103 PFU/ml to 1.2 × 104 PFU/ml. The minimal quantity of MARV virions that is detected The minimal quantity of the MARV virions that can be by RT-PCR depends on the various factors such as RNA- detected by the Ag-capture ELISA was compared with purification methods, RT-PCR methods, PCR machines, that detected by RT-PCR. The partial L-gene of MARV was visualization of the PCR products, etc., suggesting that the amplified by RT-PCR using the primer set of “Filo-A (5´- sensitivity of the Ag-capture ELISA cannot simply be com- ATCGGAATTTTTCTTTCTCATT-3´” and “Filo-B (5´- pared with that of RT-PCR. Although the RT-PCR assays, ATGTGGTGGGTTATAATAATCACTGACATG-3´)” as re- especially the nested RT-PCR, are useful, false-positive ported previously (10). Briefly, 100 μl of MARV solution and false-negative results must always be excluded. The serially diluted with human serum was treated with a QIAGEN data in the present study showing that the sensitivity of the Ag- viral RNA purification kit according to the manufacturer’s capture ELISA was almost similar to that of the RT- instructions for purification of MARV RNA. Ready-to-Go PCR suggest that a combination of the RT-PCR with the RT-PCR BeadsTM (GE Healthcare Bio-Sciences, Piscataway, Ag-capture ELISA make diagnosis of MHF more reliable. N.J., USA) was used for RT-PCR reaction. Five of 65 μl of Antigen-detection ELISA has been used as a diagnostic pro- the purified RNA solution was added to a PCR tube as a cedure for hemorrhagic fever (11,12) but not for MHF. template as well as 50 pmole of each primer and water, the To control outbreaks of hemorrhagic fever including MHF, final volume of the reaction mixture being 50 μl per tube. rapid and accurate diagnosis is essential. The newly devel- The conditions of RT-PCR were as follows: 42°C for 30 min, oped MARV nucleoprotein-capture ELISA should be further

Fig. 1. Relationship between the OD values and the dilutions of MARV solution in the Ag-capture ELISA. The ELISA plates were coated with either of the monoclonal antibodies 2A7 (A) and 2H6 (B). X-axis indicates dilution level of MARV solution (2.5× 107 PFU/ml). The figures “3”, “5”, “7”, “9”, “11”, “13” and “15” on the X-axis indicates the MARV concentration of 2.5 × 107 PFU/ml divided by 23, 25, 27, 29, 211, 213 and 215, respectively. “No-antigen” on the X-axis indicates the wells that were inoculated with samples that did not contain MARV virions. Y-axis indicates the monoclonal antibody quantity (ng/100 μl/well). Z-axis indicates the OD values. Results of the RT-PCR using the primer set of Filo-A and Filo-B is shown (C). “M” indicates “100 bp ladder DNA-size marker”. The lanes 1, 2, 3, 4 and 5 indicate the MARV virus solution diluted with human sera at 1:101, 1:102, 1:103, 1:104 and 1:105, respectively, indicating that MARV-concentration of these solutions were 2.5×106, 2.5×105, 2.5×104, 2.5 × 103 and 2.5 × 102 PFU/ml, respectively. The negative control sample did not produce any bands (data not shown).

324 evaluated for its efficacy using clinical samples collected from logic investigation of disease, Southern patients with or without MARV infections. Africa, 1975. Am. J. Trop. Med. Hyg., 27, 1210-1215. In summary, the MARV nucleoprotein-detection ELISA 6. Johnson, E.D., Johnson, B.K., Silverstein, D., Tukei, P., using monoclonal antibodies 2A7 and 2H6 detected the Geisbert, T.W., Sanchez, A.N. and Jahrling, P.B. (1996): authentic MARV nucleoprotein, suggesting that the Ag- Characterization of a new Marburg virus isolated from a capture ELISA is applicable to the diagnosis of MHF. 1987 fatal case in Kenya. Arch. Virol., Suppl. 11, 101- 114. 7. Smith, D.H., Johnson, B.K., Isaacson, M., Swanapoel, ACKNOWLEDGMENTS R., Johnson, K.M., Killey, M., Bagshawe, A., Siongok, We thank all staff members of the Unit of Biology of Viral T. and Keruga, W.K. (1982): Marburg-virus disease in Emerging Infections, Institute Pasteur, and Laboratory P4 Kenya. Lancet, 1, 816-820. Jean-Merieux-Inserm, Lyon, France, who assisted us in con- 8. Saijo, M., Niikura, M., Maeda, A., Sata, T., Kurata, T., ducting the experiments in the present study. We thank Ms. Kurane, I. and Morikawa, S. (2005): Characterization of M. Ogata, Department of Virology 1, National Institute of monoclonal antibodies to Marburg virus nucleoprotein Infectious Diseases, for her technical and clerical assistance. (NP) that can be used for NP-capture enzyme-linked This work was financially supported by grants-in-aid from immunosorbent assay. J. Med. Virol., 76, 111-118. the Ministry of Health, Labour and Welfare of Japan and by 9. Saijo, M., Niikura, M., Morikawa, S., Ksiazek, T.G., the Human Science Research Foundation, Tokyo, Japan. Meyer, R.F., Peters, C.J. and Kurane, I. (2001): Enzyme- linked immunosorbent assays for detection of anti- bodies to Ebola and Marburg using recombinant REFERENCES nucleoproteins. J. Clin. Microbiol., 39, 1-7. 1. Bausch, D.G., Borchert, M., Grein, T., Roth, C., 10. Sanchez, A., Ksiazek, T.G., Rollin, P.E., Miranda, M.E., Swanepoel, R., Libande, M.L., Talarmin, A., Bertherat, Trappier, S.G., Khan, A.S., Peters, C.J. and Nichol, S.T. E., Muyembe-Tamfum, J.J., Tugume, B., Colebunders, (1999): Detection and molecular characterization of R., Konde, K.M., Pirad, P., Olinda, L.L., Rodier, G.R., Ebola viruses causing disease in human and nonhuman Campbell, P., Tomori, O., Ksiazek, T.G. and Rollin, P.E. . J. Infect. Dis., 179 Suppl. 1, S164-169. (2003): Risk factors for Marburg hemorrhagic fever, 11.Towner, J.S., Rollin, P.E., Bausch, D.G., Sanchez, A., Democratic Republic of the Congo. Emerg. Infect. Dis., Crary, S.M., Vincent, M., Lee, W.F., Spiropoulou, C.F., 9, 1531-1537. Ksiazek, T.G., Lukwiya, M., Kaducu, F., Downing, 2. Feldmann, H., Volchkov, V.E., Volchkova, V.A. and R. and Nichol, S.T. (2004): Rapid diagnosis of Ebola Klenk, H.D. (1999): The glycoproteins of Marburg and hemorrhagic fever by reverse transcription-PCR in an Ebola virus and their potential roles in pathogenesis. outbreak setting and assessment of patient viral load as a Arch. Virol., Suppl. 15, 159-169. predictor of outcome. J. Virol., 78, 4330-4341. 3. Feldmann, H. and Klenk, H.D. (1996): Marburg and 12. Ksiazek, T.G., Rollin, P.E., Williams, A.J., Bressler, Ebola viruses. Adv. Virus Res., 47, 1-52. D.S., Martin, M.L., Swanepoel, R., Burt, F.J., Leman, 4. Martini, G.A., Knauff, H.G., Schmidt, H.A., Mayer, G. P.A., Khan, A.S., Rowe, A.K., Mukunu, R., Sanchez, and Baltzer, G. (1968): [On the hitherto unknown, in A. and Peters, C.J. (1999): Clinical virology of Ebola monkeys originating infectious disease: Marburg virus hemorrhagic fever (EHF): virus, virus antigen, and IgG disease]. Dtsch. Med. Wochenschr., 93, 559-571. and IgM antibody findings among EHF patients in 5. Conrad, J.L., Isaacson, M., Smith, E.B., Wulff, H., Crees, Kikwit, Democratic Republic of the Congo, 1995. J. M., Geldenhuys, P. and Johnston, J. (1978): Epidemio- Infect. Dis., 179, Suppl. 1, S177-187.

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