title: Development of a measles specific IgM ELISA for use with serum and oral fluid samples using recombinant measles nucleoprotein produced in Saccharomyces cerevisiae.
abstract: In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein. Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter. High levels of rMVN (20 mg/litre of yeast culture) were generated. Electron microscopy showed that the purified rMVN assembled into typical herring-bone structures. Monoclonal antibodies produced to the rMVN also reacted with native measles virus N in immunofluorescence tests. The purified rMVN and a monoclonal antibody to the rMVN conjugated to horseradish peroxidase were used to develop a measles specific IgM capture EIA (MACEIA) in both serum and oral fluid specimens. Evaluations of the MACEIA were performed by testing a) serum samples (n=80) and b) paired oral fluid/serum samples from measles cases (n=50, representing 16 cases) and oral fluids from controls with non-measles rash (n=59, representing 48 cases). The samples were also tested for measles IgM, using a reference radioimmunoassay (MACRIA). The sensitivity and specificity of the MACEIA compared with MACRIA for a) the serum samples were 100 and 96.6% respectively and b) for paired serum/oral fluids samples 100 and 100%, respectively.
authors.affiliation: Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK. dsamuel@phls.nhs.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: Alma
authors.initials: A
authors.lastname: Gedvilaite
authors.forename: Rimas
authors.initials: R
authors.lastname: Slibinskas
authors.forename: Stuart
authors.initials: S
authors.lastname: Beard
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
authors.forename: Solange Artimos
authors.initials: SA
authors.lastname: Oliveira
authors.forename: Juozas
authors.initials: J
authors.lastname: Staniulis
authors.forename: Bernard
authors.initials: B
authors.lastname: Cohen
authors.forename: David
authors.initials: D
authors.lastname: Brown
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007075:Immunoglobulin M; D008457:Measles / Q000175:diagnosis; D008459:Measles virus / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009698:Nucleoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012463:Saliva / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D007075:Immunoglobulin M; D009698:Nucleoproteins; D011994:Recombinant Proteins