Protocol: PK Bridging ELISA for Use with Anti-YTE Monoclonal Antibodies

Protocol: PK Bridging ELISA for Use with Anti-YTE Monoclonal Antibodies

Pharmacokinetic (PK) Bridging ELISA: For Use with Anti-YTE Monoclonal Antibodies Catalog TZA0135 with TZA0136/TZA0136P

This method provides a procedure for carrying out a PK ELISA with Anti-YTE Antibodies, TZA0135 (capture antibody) and HRP-conjugated TZA0136/TZA0136P (detection antibody), using antibodies with YTE mutations for the standard curve (e.g., Recombinant Human IgG1 YTE Kappa Allotype G1m3, HCA413). Please note that we recommend using our alternative PK pair TZA0137 (capture antibody) and TZA0137P (detection antibody) for drugs that have other mutations in their Fc region in addition to the YTE mutations. 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)
Phosphate buffered saline (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)
QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
Recombinant Human IgG1 YTE Kappa Allotype G1m3 (HCA413)

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. If using TZA0136P (AbD64789pap) for detection, proceed to step 2. If using TZA0136 (AbD64789ad) for detection, prepare the detection anti-YTE antibody: couple TZA0136 to a suitable SpyCatcher Reagent (e.g., TZC002P) using the (Bi)SpyCatcher coupling protocol. Please refer to the SpyCatcher-Coupling Calculator for support in performing the (Bi)SpyCatcher coupling protocol.
  2. Prepare the capture Anti-YTE Antibody TZA0135 (AbD64785ad) 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, and incubate overnight at 4°C.
  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 a dilution series of antibody with YTE mutation in 10% human serum in PBST in triplicate. Final concentration of the YTE-mutated antibody should range from 0.008 ng/mL to 32,000 ng/mL. Include a zero YTE concentration as the background value. Recombinant Human IgG1 YTE Kappa Allotype G1m3 (HCA413) can be used as a positive control for the assay.
  7. Add 20 µL of YTE-mutated antibody 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.
  8. Wash the microtiter plate 5x with PBST.
  9. To each well, add 20 µL HRP-conjugated detection Anti-YTE Antibody, TZA0136 (AbD64789ad) or TZA0136P (AbD64789pap), at 0.5 µg/mL in HISPEC Assay Diluent. Incubate for 1 hr at RT. 
  10. Wash the microtiter plate 10x with PBST.
  11. Add 20 µL QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.