Protocol of Pharmacokinetic (PK) Antigen Capture ELISA Measuring Bound Drug Exclusively

To support preclinical/clinical pharmacokinetic (PK) studies, Creative Biolabs presents a simple yet comprehensive protocol of PK antigen capture ELISA measuring bound drug exclusively in tested sample.

Notes:

1. This method should always be used in conjunction with product and batch specific information provided with each vial (see product datasheets).
2. This protocol will also need to be adjusted for use with different detection methods and immunoassay technology platforms if required.

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 human 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 antigen. 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 antibody reference in 10% human serum in PBST in triplicate. Final concentrations of antibody reference should cover the range from 0.1 ng/ml to 1,000 ng/ml. A zero antibody reference concentration as the background value is needed.
6. Add 20 μl of each of the diluted antibody 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 pre-prepared HRP conjugated detection antibody at 2 µg/ml in dilution buffer. Incubate for 1 hour at RT.
9. Wash the microtiter plate ten times with PBST.
10. Add 20 μl substrate to each well and measure the fluorescence after 30 minutes.
11. Data analysis.

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