The reference sera were analyzed for antibodies against selected HHV antigens (Table 1 ) by species-specific Monoplex and Multiplex Serology, as described previously [19 (link)]. Briefly, HHV GST-tag fusion proteins were loaded onto glutathione casein-coated fluorescence-labelled polystyrene beads (COOH-beads xMAP Technology Microspheres, Luminex Corp. Austin, Texas, USA) by in situ affinity purification from lysate. Up to 100 bead sets are distinguishable by the Luminex flow cytometer via different ratios of two fluorescent dyes within the polystyrene microspheres. Loading each antigen onto a specific bead set enables simultaneous measurement of antibodies against different antigens within one reaction vessel.
Detection of bound primary antibodies from serum took place with a biotinlyated goat-α-human IgM/IgG/IgA secondary antibody (1:1000, #109-065-064, Jackson Immunoresearch, West Grove, PA, USA) and subsequent incubation with streptavidin-R-phycoerythrin (1:750, PE-Streptavidin Conjugate, MOSS Inc., Pasadena, CA, USA) as reporter dye. Median Fluorescence Intensities (MFI) from at least 100 detected beads per bead set (e.g. antigen) were calculated for each serum. Monoplex Serology was conducted for each HHV species-specific assay in an individual experiment only comprising the species-specific antigens and GST-tag antigen for background subtraction in dilutions 1:100 and 1:1000. Optimal serum dilution was 1:1000 with the exception of VZV (1:100). In addition, performance of HHV Monoplex Serology assays were assessed in multiplex format by combining them with various pathogen-specific Monoplex Serology assays (e.g. human herpes viruses 6–8, human polyomaviruses, human papillomaviruses, human hepatitis B and C viruses) into a Multiplex Serology panel.
Detection of bound primary antibodies from serum took place with a biotinlyated goat-α-human IgM/IgG/IgA secondary antibody (1:1000, #109-065-064, Jackson Immunoresearch, West Grove, PA, USA) and subsequent incubation with streptavidin-R-phycoerythrin (1:750, PE-Streptavidin Conjugate, MOSS Inc., Pasadena, CA, USA) as reporter dye. Median Fluorescence Intensities (MFI) from at least 100 detected beads per bead set (e.g. antigen) were calculated for each serum. Monoplex Serology was conducted for each HHV species-specific assay in an individual experiment only comprising the species-specific antigens and GST-tag antigen for background subtraction in dilutions 1:100 and 1:1000. Optimal serum dilution was 1:1000 with the exception of VZV (1:100). In addition, performance of HHV Monoplex Serology assays were assessed in multiplex format by combining them with various pathogen-specific Monoplex Serology assays (e.g. human herpes viruses 6–8, human polyomaviruses, human papillomaviruses, human hepatitis B and C viruses) into a Multiplex Serology panel.
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