Protocol of Pharmacokinetic (PK) Bridging ELISA Measuring Free Drug

This protocol represents an example of pharmacokinetic (PK) bridging ELISA measuring free drug in the sample. For total drug or bound drug measurement, see the sections entitled Pharmacokinetic (PK) Bridging ELISA Measuring Total Drug and Pharmacokinetic (PK) Antigen Capture ELISA Measuring Bound Drug Exclusively.

Reagents

• PBS: 136 mM NaCl

  2.68 mM KCl

  8.1 mM Na₂HPO₄

  1.46 mM KH₂PO₄

• PBST: PBS with 0.05% Tween-20
• BSA
• Human Serum
• Fluorogenic Peroxidase Substrate

Materials

• 384-well microtiter plate, black, square flat-bottom wells.
• Qualified 96-well plates can be used instead of 384-well plates. For the 96-well format, use 100 μl (instead of 20 μl) of antigen, antibodies, or substrate and 300 μl for the blocking step.
• Fluorescence plate reader.

Method

1. Prepare the anti-Drug A capture antibody 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 Drug A in 10% human serum in PBST in triplicate. Final concentrations of Drug A should cover the range from 0.1 ng/ml to 1,000 ng/ml. Include a zero Drug A concentration as the background value.
6. Add 20 μl of each of the diluted Drug A 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 anti-Drug A antibody at 2 µg/ml (diluted in appropriate buffer). Incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. Add 20 μl fluorogenic peroxidase substrate to each well and measure the fluorescence after 30 minutes.
11. Data analysis.

Notes:

1. For the convenience of description, Drug A represents the antibody drug to be tested in sample.
2. For step 4, standard concentrations can be set to cover a wider range, from 0.1 ng/ml to 8,000 ng/ml depending on the drug to be tested.

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