title: Spectrally and Spatially Multiplexed Serological Array-in-Well Assay Utilizing Two-Color Upconversion Luminescence Imaging.
abstract: We demonstrate a simple dual-mode multiplexed array-in-well immunoassay for simultaneous classification and detection of serum IgG and IgM antibodies against influenza A and human adenoviruses based on the color and position of the upconversion luminescence on the array. Biotinylated influenza A/H1N1 and A/H5N1 as well as adenovirus serotype 2 and 5 hexon antigens along with positive and negative controls were printed in an array format onto the bottom of streptavidin-coated microtiter wells. The anti-influenza A and antiadenovirus antibodies present in the sample were captured to the array and detected with antihuman antibody-coated upconverting nanophosphors (UCNPs). The green emitting UCNPs (NaYF4:Yb(3+),Er(3+)) coated with antihuman IgG and blue emitting UCNPs (NaYF4:Yb(3+),Tm(3+)) coated with antihuman IgM were used to detect human IgG and IgM antibodies, respectively. The emission of the bound UCNPs was imaged free of autofluorescence with anti-Stokes photoluminescence microwell imager. No spectral cross-talk was observed between green and blue emitting UCNPs. Also the cross-reactivities between UCNP-conjugates and immobilized human IgG and IgM antibodies were negligible. Position of the signal on the array defined the antigen specificity and the antibody class was defined by the color of the upconversion luminescence. This technology could be used for differentiation between acute infection from past infection and immunity. Additionally, the class of the antibody response can be used for the differentiation between primary and secondary infections, hence, facilitating epidemiological seroprevalence studies.
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Vishal
authors.initials: V
authors.lastname: Kale
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Henna
authors.initials: H
authors.lastname: Päkkilä
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Jiri
authors.initials: J
authors.lastname: Vainio
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Anna
authors.initials: A
authors.lastname: Ahomaa
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Nina
authors.initials: N
authors.lastname: Sirkka
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Annika
authors.initials: A
authors.lastname: Lyytikäinen
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Sheikh Mohammad
authors.initials: SM
authors.lastname: Talha
authors.affiliation: Department of Virology, University of Turku , Kiinamyllynkatu 13, FI-20520 Turku, Finland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kutsaya
authors.affiliation: Department of Virology, University of Turku , Kiinamyllynkatu 13, FI-20520 Turku, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Waris
authors.affiliation: Department of Virology, University of Turku , Kiinamyllynkatu 13, FI-20520 Turku, Finland.|Virology Unit, National Institute for Health and Welfare , Helsinki, Finland.
authors.forename: Ilkka
authors.initials: I
authors.lastname: Julkunen
authors.affiliation: Department of Biotechnology, University of Turku , Tykistökatu 6 A, FI-20520 Turku, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D000256:Adenoviridae / Q000276:immunology; D000937:Antigen-Antibody Reactions; D003116:Color; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000097:blood* / Q000276:immunology; D007075:Immunoglobulin M / Q000097:blood* / Q000276:immunology; D009980:Influenza A virus / Q000276:immunology; D049449:Luminescence; D012698:Serologic Tests
chemical_list: D007074:Immunoglobulin G; D007075:Immunoglobulin M