Protocol: PK ELISA Antigen Capture Format For Use With Anti-Adalimumab Antibody

Protocol: PK ELISA Antigen Capture Format For Use With Anti-Adalimumab Antibody

Protocol: PK ELISA Antigen Capture Format For Use With Anti-Adalimumab Antibody

PK ELISA Antigen Capture Format For Use With Anti-Adalimumab Antibody

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Pharmacokinetic (PK) ELISA Antigen Capture Format: For use with anti-adalimumab monoclonal antibody products HCA231 or HCA232/HCA232P

This method provides a procedure for carrying out a PK ELISA Antigen Capture Format with anti-adalimumab antibodies, product codes HCA231 or HCA232P (detection antibody), and using adalimumab monoclonal antibody for the standard curve.

Anti-adalimumab drug/target complex antibody recognizes adalimumab only when bound to its target TNFα. It does not recognize the free drug or unbound 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.


Reagents

BSA
HISPEC immunoassay diluent (Bio-Rad, BUF049)
Human Serum (Sigma-Aldrich, H4522)
Lynx Rapid HRP Antibody Conjugation Kit® (Bio-Rad, LNK001P-LNK006P)
For best results when conjugating with Lynx Rapid HRP Antibody Conjugation Kit, avoid using antibody with thiomersal as preservative. Contact us to discuss thiomersal-free options. 
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)
Recombinant human TNFα (Bio-Rad, PHP051)

Materials

384-well microtiter plate, black, square flat-bottom wells, MaxiSorp™ PS (Thermo Fisher Scientific, 460518)
Fluorescence plate reader
96-well plates can be used instead of 384-well plates, e.g. black, flat-bottom MaxiSorp PS (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 detection antibody: if using HCA231 (AbD20350) for detection, first conjugate the antibody using a Lynx Rapid HRP Antibody Conjugation Kit;  HCA232P (AbD20349_hIgG1) is already directly labeled with HRP.
  2. Prepare human TNFα (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. Incubate overnight at 4°C.
  3. Wash the microtiter plate five times with PBST.
  4. Block the microtiter plate by adding 100 μl 5% BSA in PBST to each well, and then incubate for 1 hour at room temperature (RT).
  5. Wash the microtiter plate five times with PBST.
  6. For the standard curve, prepare a dilution series of adalimumab in 10% human serum in PBST in triplicate. Final concentrations of adalimumab should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero adalimumab concentration as the background value.
  7. Add 20 μl of each of the diluted standards to the wells designated for the standard curve (in triplicate for each standard recommended). Add 20μl of each test sample to the other wells (in triplicate for each sample recommended). Incubate for 1 hour at RT.
  8. Wash the microtiter plate five times with PBST.
  9. To each well, add 20 µl pre-prepared HRP conjugated detection antibody at 2 µg/ml in HISPEC buffer. Incubate for 1 hour at RT.
  10. Wash the microtiter plate ten times with PBST.
  11. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V4.10.2015


Lynx Rapid Conjugation Kit® is a registered trademark of Bio-Rad AbD Serotec Ltd.
MaxiSorp™ and QuantaBlu™ are trademarks of Thermo Fisher Scientific.
Tween® is a registered trademark of ICI Americas Inc.