Protocol: PK Bridging ELISA For Use With Anti-Adalimumab Antibody

Protocol: ADA Bridging ELISA for use with Anti-Etanercept Antibody

Protocol: ADA Bridging ELISA For Use With Anti-Adalimumab Antibody

Protocol: ADA Bridging ELISA for use with Anti-Etanercept Antibody

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Anti-Drug Antibody (ADA) Bridging ELISA: for use with Anti-Etanercept Antibody product HCA277

This method provides a procedure for generating an ADA ELISA standard curve with Anti-Etanercept Antibody, catalog number HCA277, using etanercept for capture and detection. 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 Assay Diluent (catalog #BUF049)
Human Serum (Sigma-Aldrich, catalog #H4522)
LYNX Rapid HRP Antibody Conjugation Kit® (catalog #LNK001P-LNK006P)
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, catalog #15169)

Materials

384-well microtiter plate, black, square flat-bottom wells, e.g. Black 384-Well Immuno Plates (Thermo Fisher Scientific, catalog #460518)
Fluorescence plate reader
96-well plates can be used instead of 384-well plates (black, flat-bottom wells) e.g. Black 96-Well Immuno Plates (Thermo Fisher Scientific, catalog #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: conjugate etanercept using a LYNX Rapid HRP Antibody Conjugation Kit.
  2. Prepare unconjugated etanercept 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 etanercept solution, and 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 Anti-Etanercept Antibody, catalog #HCA277 (clone AbD25940_hIgG4Pro) in 10% human serum in PBST in triplicate. Final concentration of anti-etanercept antibody should cover the range from 1 ng/ml to 32,000 ng/ml. Include a zero anti-etanercept antibody concentration as the background value.
  7. Add 20 µl of anti-etanercept antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hour at RT.
  8. Wash the microtiter plate five times with PBST.
  9. To each well, add 20 µl HRP conjugated etanercept diluted to 2 µg/ml in HISPEC Assay Diluent and incubate for 1 hour at RT.
  10. Wash the microtiter plate ten times with PBST.
  11. Add 20 μl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 minutes.