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

Protocol: PK Bridging ELISA For Use With Anti-Rituximab Antibodies

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

Protocol: PK Bridging ELISA For Use With Anti-Rituximab Antibodies

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Pharmacokinetic (PK) Bridging ELISA: For use with anti-rituximab monoclonal antibody products HCA186 and MCA2260P

This method provides a procedure for carrying out a PK ELISA with anti-rituximab antibodies, product codes HCA186 (capture antibody) and MCA2260P (detection antibody), and using rituximab monoclonal antibody 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.


Reagents

BSA
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
QuantaBlu™ fluorogenic peroxidase substrate (Thermo Fisher Scientific, 15169)

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 the anti-rituximab capture antibody HCA186 (AbD18423) 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. Incubate overnight at 4°C.
  2. Wash the microtiter plate five times with PBST.
  3. 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).
  4. Wash the microtiter plate five times with PBST.
  5. For the standard curve, prepare a dilution series of rituximab in 10% human serum in PBST in triplicate. Final concentrations of rituximab should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero rituximab concentration as the background value.
  6. Add 20 μl of each of the diluted standards to the wells designated for the standard curve (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 hour at RT.
  7. Wash the microtiter plate five times with PBST.
  8. To each well, add 20 µl HRP conjugated detection antibody MCA2260P (MB2A4) at 4 µg/ml in HISPEC buffer. Incubate for 1 hour at RT.
  9. Wash the microtiter plate ten times with PBST.
  10. Add 20 μl QuantaBlu to each well and measure the fluorescence after 30 minutes.

V4.10.2015


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