Goat anti human igg fc gamma specific hrp

To test total IgG from plasma, 384‐well HB plates (Perkin Elmer) were coated with AffiniPure Goat anti‐human IgG, Fc gamma fragment antibody, at 1 μg/ml in PBS for 2 h at 37°C. To test anti‐PrP autoantibodies, plates were coated with human recPrP_23‐230. After washing and blocking in 5% Milk in PBS‐T for 90 min at RT, serial dilutions of patient plasma (starting from 1:8,000 and 1:50 dilution for total IgG and anti‐PrP autoantibody ELISA, respectively) were incubated for 2 h at 37°C. Pooled normal human plasma (Innovative Research, #IPLA‐N), IgG‐Kappa, IgG‐Lambda at a concentration of 200 ng/ml or humanized POM1, (hPOM1 at 200 ng/ml and 2 μg/ml for total IgG and anti‐PrP autoantibody ELISA, respectively) served as controls. After washing five times, the following HRP‐coupled secondary antibodies were incubated at 1:4,000 in PBS‐T for 1 h, at RT: Goat anti‐human Kappa‐HRP, goat anti‐human Lambda‐HRP or goat anti‐human IgG Fc‐gamma specific‐HRP (Jackson ImmunoResearch #109‐035‐098). The plates were developed as described above.

For further validation, 50–250 μl of plasma were used for immunoglobulin purification via 1:1 mixture of proteinA/proteinG resin (GE Healthcare). Slurry was washed once with PBS. Blood samples were spun down at 16,000 g for 5 min, mixed with 10% of slurry and incubated at 4°C o/n on a rotating wheel. Slurry‐blood mix was transferred to screw cap spin columns (Pierce) and beads were washed twice with PBS. Beads were spun down one more time to remove residual PBS and 100 μl of 0.1 M glycine, pH 1.8 was added to the beads. Four hundred microliter of 1 M Tris, pH 8.0 were added to the bottom of the spin columns for neutralization of elution buffer after centrifugation. For buffer exchange, 0.5 ml Centrifugal Filters (Amicon) were used. Protein concentration was measured using NanoDrop ND‐1000 Spectrophotometer.
IgG fractions of blood samples were diluted at 100 μg/ml in 1% skim milk in PBS‐T and assessed for binding to human recPrP_23‐230, murine recPrP_23‐110 and human recPrP_121‐230 and BSA (coated at 43 nM) in ELISA using goat anti‐human IgG Fc‐gamma specific‐HRP (Jackson ImmunoResearch #109‐035‐098), 1:4,000 for detection.