title: Simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay.
abstract: We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
authors.affiliation: Laboratory Animal Resource Center, University of Tsukuba, Ibaraki 305-8575, Japan. kunitasa@md.tsukuba.ac.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kunita
authors.forename: Kanako
authors.initials: K
authors.lastname: Kato
authors.forename: Miyuki
authors.initials: M
authors.lastname: Ishida
authors.forename: Kozue
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Shuko
authors.initials: S
authors.lastname: Kameda
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ishida
authors.forename: Akira
authors.initials: A
authors.lastname: Takakura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Goto
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Sugiyama
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yagami
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D019411:Clinical Laboratory Techniques / Q000379:methods*; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D005260:Female; D005453:Fluorescence; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000276:immunology*; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D012376:Rodent Diseases / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000914:Antibodies, Viral; D012367:RNA, Viral