PK Bridging ELISA Detecting Total Drug using Anti-Golimumab Antibodies
Pharmacokinetic (PK) bridging ELISA protocol, detects total drug (free, partially and fully bound): for use with Anti-Golimumab Monoclonal Antibodies HCA289 and HCA290P
This method provides a procedure for carrying out a PK ELISA with Anti-Golimumab Antibodies, HCA289 (capture antibody) and HCA290P (detection antibody), and using golimumab for the standard curve. The method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets). This protocol will need to be adjusted for use with different detection methods and immunoassay technology platforms.
View all of our Golimumab antibodiesReagents
- BSA
HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522) - 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
- Prepare the Anti-Golimumab Capture Antibody, HCA289 (AbD25455) 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.
- Wash the microtiter plate five times (5x) with PBST.
- Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
- Wash the microtiter plate 5x with PBST.
- For the standard curve, prepare a dilution series of golimumab in 10% human serum in PBST in triplicate. Final concentration of golimumab should cover the range from 0.1 ng/ml to 8,000 ng/ml. Include a zero golimumab concentration as the background value.
- Add 20 µl of golimumab 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.
- Wash the microtiter plate 5x with PBST.
- To each well, add 20 µl HRP conjugated Anti-Golimumab Detection Antibody, HCA290P (AbD25451_hIgG1) at 2µg/ml in HISPEC Assay Diluent and incubate for 1 hr at RT.
- Wash the microtiter plate 10x with PBST.
- Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.
