| Project/Area Number |
17591065
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | Hokkaido University |
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Principal Investigator |
ISHIGURO Nobuhisa Hokkaido Univ., Hospital, Infection Control Team, Research Associate, 大学病院, 助手 (40168216)
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| Co-Investigator(Kenkyū-buntansha) |
SEYA Tsukasa Hokkaido Univ., Grad.School of Medicine Dept.of Microbiology and Immunology, Professor, 大学院医学研究科, 教授 (10301805)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | human metapneumovirus / surface proteins / chromatographic immunoassa / monoclonal antobody / 迅速診断 / 液性免疫応答 |
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| Research Abstract |
A new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing human metapneumovirus (hMPV) F protein (Bac-F IFA) was developed. The majority of the antibodies detected by the hMPV IFA reacted with the hMPV fusion (F) protein. The Bac-F IFA was more sensitive than the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA).
Specific antibodies against nucleocapsid (N) and matrix (M) proteins in serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1%, N protein; 40.8%, M protein) than those against whole proteins of hMPV detected by hMPV IFA.
The monoclonal antibodies (MAbs), designated 1G3 and 9B10, against hMPV F protein were developed. These MAbs had neutralizing activity. These indicate that the hMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection.
Two mouse monoclonal antibodies (MAbs), designated 3D1 and 5B10, against N protein of hMPV were developed. A chromatographic immunoassay (lateral flow assay) was developed using the MAbs. The assay is a sandwich immunoassay that uses a paper membrane with a gold colloid-conjugated MAb (5B10) in a liquid phase and an MAb (3D1) in a solid phase. The assay had good specificity and sufficient sensitivity to detect hMPV. Therefore, the assay may be a rapid and useful test for diagnosis of hMPV infections.
An indirect immunofluorescent-antibody test (IFA) with a monoclonal antibody against hMPV was developed for detection of hMPV in nasal secretions from patients with respiratory tract infections. IFA results were positive for 11 of the 15 RT-PCR-positive children (sensitivity, 73.3%) and 1 of the 33 RT-PCR-negative children (specificity, 97.0%).
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