Protocol: PK Bridging ELISA for Use with Anti-Denosumab Antibodies

Reagents

BSA

HISPEC Assay Diluent  (BUF049)

Human Serum (Sigma-Aldrich, H4522)

Phosphate buffered saline (PBS)

136 mM NaCl
2.68 mM KCl
8.1 mM Na₂HPO₄
1.46 mM KH₂PO₄

PBST

PBS with 0.05% Tween 20

QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)

Materials

384-well microtiter plate, black, square flat-bottom wells,for example, Black 384-Well Immuno Plates (Thermo Fisher Scientific, 460518)

Fluorescence plate reader

96-well plates can be used instead of 384-well plates, (black, flat-bottom wells) for example, Black 96-Well Immuno Plates, (Thermo Fisher Scientific, 437111). For the 96-well format, use 100 µl (instead of 20 µl) of antigen, antibodies, or substrate and 300 µl for the blocking step.

Method

1. Prepare the capture Anti-Denosumab Antibody, HCA288 (AbD26296), at 1 µg/ml in PBS. Coat the required number of wells of a 384-well microtiter plate with 20 µl per well of the prepared capture antibody, and incubate overnight at 4°C.
2. Wash the microtiter plate five times (5x) with PBST.
3. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
4. Wash the microtiter plate 5x with PBST.
5. For the standard curve, prepare a dilution series of denosumab in 10% human serum in PBST in triplicate. Final concentration of denosumab should cover the range from 0.125 ng/ml to 8,000 ng/ml. Include a zero denosumab concentration as the background value.
6. Add 20 µl of denosumab dilution per well (in triplicate for each standard recommended). Add 20 µl of each test sample to the other wells (in triplicate for each sample recommended). Incubate for 1 hr at RT.
7. Wash the microtiter plate 5x with PBST.
8. To each well, add 20 µl HRP conjugated detection Anti-Denosumab Antibody, HCA280P (AbD26295_hIgG1), at 2 µg/ml in HISPEC Assay Diluent. Incubate for 1 hr at RT.
9. Wash the microtiter plate 10x with PBST.
10. Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.