title: High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology.
abstract: To develop improved reagents for mumps serology a high-level yeast expression system was employed to produce recombinant mumps nucleoprotein (rNP). The rNP was purified by CsCl gradient centrifugation and yielded approximately 15 mg/l of yeast culture. Electron microscopy of the rNP revealed characteristic herring-bone structures. The electrophoretic mobility of rNP in yeast cells was similar to native NP in SDS-PAGE. Monoclonal antibodies to rNP reacted with native mumps virus nucleoprotein by immunofluorescence assay. A monoclonal antibody to native mumps virus NP reacted with rNP by Western blot assay. The rNP was investigated as antigen in an IgM capture enzyme immunoassay (EIA) using a horseradish peroxidase conjugate of monoclonal antibody to the rNP. Eighteen sera previously found to be positive by IgM capture radioimmunoassay (MACRIA) and 30 sera that were mumps IgM negative by MACRIA were tested by mumps IgM capture EIA. The results for the two test were concordant. In addition, 26 rheumatoid factor positive sera and 35 sera that were IgM positive for measles, rubella or parvovirus B19 were tested. Fifty-nine sera were negative by mumps IgM capture EIA but two sera collected from two infants 3 and 6 weeks after mumps, measles and rubella vaccination were positive. Mumps MACRIA confirmed these results. Compared to MACRIA the overall sensitivity was 100% (20/20) and specificity was 96.8% (30/31). The yeast expressed rNP was highly immunogenic and suitable for use in IgM capture EIA for the diagnosis of mumps.
authors.affiliation: Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, London, United Kingdom. dsamuel@phls.org.uk
authors.forename: Dhanraj
authors.initials: D
authors.lastname: Samuel
authors.forename: Kestutis
authors.initials: K
authors.lastname: Sasnauskas
authors.forename: Li
authors.initials: L
authors.lastname: Jin
authors.forename: Stuart
authors.initials: S
authors.lastname: Beard
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.forename: Alma
authors.initials: A
authors.lastname: Gedvilaite
authors.forename: Bernard
authors.initials: B
authors.lastname: Cohen
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002522:Chlorocebus aethiops; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood*; D008854:Microscopy, Electron; D009107:Mumps / Q000175:diagnosis* / Q000821:virology; D009109:Mumps virus / Q000276:immunology*; D009698:Nucleoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells
chemical_list: D000914:Antibodies, Viral; D007075:Immunoglobulin M; D009698:Nucleoproteins; D011994:Recombinant Proteins