Detection of antibodies against human metapneumovirus by western blot using recombinant nucleocapsid and matrix proteins

Ishiguro, Nobuhisa; Ebihara, Takashi; Endo, Rika; Ma, Xiaoming; Kawai, Eri; Ishiko, Hiroaki; Kikuta, Hídeaki

doi:10.1002/jmv.20667

Cited by 8 publications

(1 citation statement)

References 24 publications

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“…Ishiguro et al tested for specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 sera by Western blot using recombinant N and M proteins of HMPV expressed in E. coli. 33 The results were compared with those of immunofluorescence assays (IFAs) based on HMPV-infected LLC-MK2 cells, which expressed the whole HMPV proteome. Their results indicated that 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 IFA.…”

Section: Discussionmentioning

confidence: 99%

Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus

O'Shaughnessy

Carr

Crowley

et al. 2011

Journal of Clinical Virology

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a b s t r a c tBackground: Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens. Objective: To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies. Study design: HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA. Results: High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N-and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1-15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested. Conclusion: This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

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Section: Discussionmentioning

confidence: 99%

Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus

O'Shaughnessy

Carr

Crowley

et al. 2011

Journal of Clinical Virology

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Human metapneumovirus infection

Warris

Groot

2007

Pediatric Infectious Diseases Revisited

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Generation of recombinant nucleocapsid protein of human metapneumovirus in baculovirus for detecting antibodies in the Beijing population

et al. 2009

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Human metapneumovirus (hMPV) is associated with respiratory infections in both adults and children. Recombinant nucleocapsid (N) proteins of hMPV from two major groups of hMPV in Beijing with a 6x His-tag at the C terminus were constructed in a baculovirus and expressed in transfected Sf21 insect cells. The antigenicity and specificity of the expressed recombinant proteins were examined by immunofluorescence assay and western blotting. Preliminary use of the expressed proteins for antibody detection in 187 serum specimens collected from different age groups in Beijing, China, indicated that the purified recombinant N-His proteins were of good antigenicity and specificity and could be a potential antigen for further seroprevalence study of hMPV, especially in the Chinese population. The positive rate for antibody detection suggested that hMPV from two clusters was co-circulating in Beijing and that most of the people in Beijing have been exposed to the virus by age 60.

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Detection of antibodies against human metapneumovirus by western blot using recombinant nucleocapsid and matrix proteins

Cited by 8 publications

References 24 publications

Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus

Recombinant expression and immunological characterisation of proteins derived from human metapneumovirus

Human metapneumovirus infection

Generation of recombinant nucleocapsid protein of human metapneumovirus in baculovirus for detecting antibodies in the Beijing population

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