Protocol: PK Antigen Capture ELISA For Use With Anti-Infliximab Antibodies
Pharmacokinetic (PK) Antigen Capture ELISA: For Use with Anti-Infliximab Monoclonal Antibody catalog TZA009
This method provides a procedure for carrying out a PK ELISA Antigen Capture Format with Anti-Infliximab Antibody, TZA009 (detection antibody), and using infliximab for the standard curve. Anti-Infliximab drug/target complex antibody recognizes infliximab only when bound to its target human TNFα. It does not recognize the free drug or unbound human TNFα. 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 infliximab antibodiesReagents
- BSA (Sigma-Aldrich, A7906-100)
HISPEC immunoassay diluent (Bio-Rad, 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 (Merck Millipore, 817072)
- QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
Recombinant Human TNFα Protein (Bio-Rad, PHP051H)
Human Anti-TNFα Antibody (Bio-Rad, MCA6090)
Mouse Anti-His Tag Antibody:HRP (Bio-Rad, #MCA5995P)
Materials
- 384-well microtiter plate, black, 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 human TNFα protein (capture antigen) at 5 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 antigen, and incubate overnight at 4°C.
- Wash the microtiter 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 infliximab in 10% human serum in PBST in triplicate. Final concentration of infliximab should cover the range from 0.001 ng/ml to 1,000 ng/ml. Include a zero infliximab concentration as the background value.
- Add 20 μl of infliximab dilution per well (in triplicate for each standard is recommended). Add 20 μl of each test sample to the other wells (in triplicate for each sample is recommended). Incubate for 1 hr at RT.
- Wash the microtiter plate 5x with PBST.
- To each well, add 20 µl Anti-infliximab Antibody, TZA009 (AbD18391ad) at 1 µg/ml in PBST. Incubate for 1 hr at RT.
- Wash the microtiter plate 5x with PBST.
- To each well, add 20 µl detection Mouse anti His tag antibody:HRP (Bio-Rad, MCA5995P) 1:2000 in HISPEC diluent (BUF049). 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.
