Modified αVIII-FLI Assay for Anti-FVIII Antibodies

The αVIII-FLI is a modified version of our previously described method(18 (link)). Briefly, plasma samples diluted 1:30 in phosphate buffered saline (PBS) containing 1% dried milk (PBSM) were incubated with SeroMAP beads (Luminex Corporation, Austin, TX) coupled to Kogenate FS (Bayer Healthcare, Tarrytown, NY). Anti-FVIII antibodies were detected using serial incubations with biotinylated anti-human Ig (anti-IgG1, A-10650; anti-IgG2, 05-3540; anti-IgG3, MH1532; anti-IgG4, A-10663; anti-IgM, H15015; Life Technologies, Carlsbad, CA) and R-phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) using a Bio-Plex 200 suspension array system (Bio-Rad Laboratories, Hercules, CA). Results are expressed as median fluorescence intensity (MFI). The threshold for positivity was set at two standard deviations above the mean MFI of the results obtained for healthy donors.

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Boylan B., Rice A.S., Dunn A.L., Tarantino M.D., Brettler D.B., Barrett J.C, & Miller C.H. (2014). Characterization of the anti-factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence-based immunoassay. Journal of thrombosis and haemostasis : JTH, 13(1), 47-53.

Publication 2014

Kogenate FS R-phycoerythrin-conjugated streptavidin Bio-Plex 200 suspension array system

Anti antibodies Anti igm Austin Donors Fluorescence Human Igg1 Igg2 Igg3 Igg4 Kogenate fs Milk Phosphate Phycoerythrin Plasma Saline Streptavidin

Corresponding Organization :

Other organizations : Centers for Disease Control and Prevention, Emory University, Bleeding & Clotting Disorders Institute, National Hemophilia Foundation, Virginia Commonwealth University

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Variable analysis

independent variables

Plasma sample dilution (1:30 in PBS containing 1% dried milk)

dependent variables

Median fluorescence intensity (MFI) of anti-FVIII antibodies

control variables

Incubation of plasma samples with SeroMAP beads coupled to Kogenate FS
Incubation with biotinylated anti-human Ig (anti-IgG1, anti-IgG2, anti-IgG3, anti-IgG4, anti-IgM)
Incubation with R-phycoerythrin-conjugated streptavidin
Use of Bio-Plex 200 suspension array system
Threshold for positivity set at two standard deviations above the mean MFI of healthy donors

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