Domestic cat microsphere immunoassays: Detection of antibodies during feline immunodeficiency virus infection

Abstract

Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9–28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases.

Pathogenesis of oral FIV infection

“…Saliva samples from FIV-infected and negative control cats were diluted 1:100 and 1:1000 in assay buffer for detection of IgG and IgA, respectively. These samples were then incubated in duplicate with approximately 2,500 conjugated beads per well in untreated, round-bottom 96-well plates [ 51 , 60 ]. Total IgG and IgA antibody concentrations were calculated from an 8-point standard curve (2-fold dilution series, run in duplicate) using the MFI obtained from ≥100 microspheres per analyte per well (Bio-Plex ™ Manager 5.0).…”

“…Total IgG and IgA antibody concentrations were calculated from an 8-point standard curve (2-fold dilution series, run in duplicate) using the MFI obtained from ≥100 microspheres per analyte per well (Bio-Plex ™ Manager 5.0). Reagent concentrations, volumes, incubation times, acceptable standard recovery, and data analysis were performed as previously described [ 51 , 60 ].…”

“…FIV-specific antibodies were detected using microsphere immunoassay (MIA) protocols involving conjugation of magnetic microspheres with FIV-specific capsid (CA) or envelope surface glycoprotein (SU C36 -Fc) recombinant proteins [ 51 , 60 ]. Following conjugation protocols, a hemocytometer was used to determine microsphere concentrations, and protein coupling was confirmed via incubation of microspheres with primary antibodies and/or PE-conjugated detection antibodies [ 51 ]. Successful coupling was determined by a median fluorescence intensity (MFI) of >2,000.…”

“…The MFI was calculated from ≥100 microspheres per analyte per well (Bio-Plex ™ Manager 5.0) and then used for data analysis. All reagent concentrations, volumes, incubation times, acceptable standard recovery, and data analysis were as previously described [ 51 , 60 ].…”

“…Saliva from naïve SPF (specific pathogen free) cats was incubated with a viral stock of FIV C36 and tested for capacity to inhibit viral growth in Crandall feline kidney (CRFK) cell cultures. Coincident studies evaluating hematologic and clinical features of infection in these same cats allowed comparisons of oral and peripheral characteristics of viral infection [ 50 , 51 ].…”