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

Reagents

BSA (Sigma-Aldrich, A7906)
HISPEC Assay Diluent  (BUF049)
Human Serum (Sigma-Aldrich, H4522)
SpyCatcher Reagents e.g. BiSpyCatcher2:HRP (TZC002P)
Contact us to discuss alternative SpyCatcher options

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 (Merck Millipore, 817072)

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, 10395991)

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-Canakinumab Antibody TZA063 (AbD54116ad) 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. If using TZA064P (AbD51668pap) for detection proceed to step 3. If using TZA064 for detection, prepare the detection anti-canakinumab antibody: couple TZA064 (AbD54331ad) to a suitable SpyCatcher Reagent (e.g. TZC002P) using the appropriate coupling protocol.  
3. Wash the microtiter plate five times (5x) with PBST.
4. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
5. Wash the microtiter plate 5x with PBST.
6. For the standard curve, prepare a dilution series of canakinumab in 10% human serum in PBST in triplicate. Final concentration of canakinumab should cover the range from 0.1 ng/ml to 10,000 ng/ml. Include a zero canakinumab concentration as the background value.
7. Add 20 µl of canakinumab 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.
8. Wash the microtiter plate 5x with PBST.
9. To each well, add 20 µl of either (Bi)SpyCatcher:HRP-coupled detection Anti-Canakinumab Antibody, TZA064 (AbD54331ad), or HRP-conjugated detection Anti-Canakunumab Antibody, TZA064P (AbD54331pap), at 0.5 µg/ml in HISPEC Assay Diluent. Incubate for 1 hr at RT.
10. Wash the microtiter plate 10x with PBST.
11. Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.