Protocol: PK Bridging ELISA for Use with Anti-Denosumab Antibodies

Protocol: PK Bridging ELISA for Use with Anti-Eculizumab Antibodies

Protocol: PK Bridging ELISA for Use with Anti-Eculizumab Antibodies

Protocol: PK Bridging ELISA for Use with Anti-Eculizumab Antibodies

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Pharmacokinetic (PK) Bridging ELISA: For Use with Anti-Eculizumab Monoclonal Antibody Catalog HCA339 and Anti-Human C5 Monoclonal Antibodies TZA034/TZA034P

This method provides a procedure for carrying out a PK ELISA with Anti-Eculizumab Drug/Target Complex Antibody, HCA339 (capture antibody) and HRP-conjugated Anti-Human C5 Antibodies TZA034/TZA034P (detection antibody) and using eculizumab for the standard curve. Anti-eculizumab drug/target complex antibody recognizes eculizumab only when bound to its target human C5. It does not recognize the free drug or unbound human C5. 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 (Sigma-Aldrich, A7906)
HISPEC Assay Diluent (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 (Merck Millipore, 817072)
SpyCatcher Reagents e.g. BiSpyCatcher2:HRP (TZC002P)
Contact us to discuss alternative SpyCatcher options
QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)

Materials

384-well microtiter plate, black, square flat-bottom wells, for example, Black 384-Well Immuno Plates (Thermo Fisher Scientific, 460518)
Fluorescence plate reader
96-well plates can be used instead of 384-well plates (black, flat-bottom wells), for example, Black 96-Well Immuno Plates (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 capture Anti-Eculizumab Drug/Target Complex Antibody HCA339 (AbD32334) 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 antibody, and incubate overnight at 4°C.
  2. If using TZA034P (AbD36700pap) for detection proceed to step 3. If using TZA034 for detection, prepare the detection Anti-Human C5 Antibody: Couple TZA034 (AbD36700ad) to a suitable SpyCatcher reagent (e.g.TZC002P) using the appropriate coupling protocol. 
  3. Wash the microtiter plate five times (5x) with PBST.
  4. Block the microtiter plate by adding 100 µl 5% BSA in PBST to each well, and then incubate for 1 hr at RT.
  5. Wash the microtiter plate 5x with PBST.
  6.  For the standard curve, prepare the drug/target complex of eculizumab and human C5 by adding increasing concentrations of eculizumab to 20% human serum (source of human C5) in PBST in triplicate. Final concentration of eculizumab should cover the range from 0.01 ng/ml to 10,000 ng/ml. Include a zero eculizumab concentration as the background value.
  7. Incubate for 30 min at RT.
  8. Add 20 µl of the drug/target complex dilution per well (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 hr at RT.
  9. Wash the microtiter plate 5x with PBST.
  10. To each well, add 20 µl of either (Bi)SpyCatcher:HRP-coupled detection Anti-Human C5 Antibody, TZA034 (AbD36700ad), or HRP-conjugated detection Anti-Human C5 Antibody, TZA034P (AbD36700pap) at 0.5 µg/ml in HISPEC Assay Diluent. Incubate for 1 hr at RT.
  11. Wash the microtiter plate 10x with PBST.
  12. Add 20 µl QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.