Protocol: PK Monomethyl auristatin E (MMAE) Conjugated Antibody

Protocol: PK Monomethyl Auristatin E (MMAE) Conjugated Antibody

Protocol: PK Monomethyl Auristatin E (MMAE) Conjugated Antibody

Protocol: PK Monomethyl Auristatin E (MMAE) Conjugated Antibody

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Pharmacokinetic (PK) Bridging ELISA: For Use with Anti-MMAE Monoclonal Antibodies Catalog TZA056 and TZA057 Coupled to a Suitable SpyCatcher Reagent (e.g. TZC002P)

This method provides a procedure for carrying out a PK ELISA with Anti-MMAE Antibodies, TZA056 (capture antibody) and HRP-conjugated TZA057 (detection antibody), using an MMAE-conjugated 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.


BSA (Sigma-Aldrich, A7906)
​HISPEC Assay Diluent (BUF049)
Human Serum (Sigma-Aldrich, H4522)
SpyCatcher Reagents e.g. BiSpyCatcher2:HRP (TZC002P)
Contact us to discuss alternative SpyCatcher options
136 mM NaCl
2.68 mM KCl
8.1 mM Na2HPO4
1.46 mM KH2PO4
PBS with 0.05% Tween 20 (Merck Millipore, 817072)
QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)


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.


  1. Prepare the capture Anti-MMAE Antibody TZA056 (AbD50347ad) 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.
  2. Prepare the detection Anti-MMAE Antibody: couple TZA057 (AbD50348ad) to a suitable SpyCatcher reagent (e.g. TZC002P) using the (Bi)SpyCatcher 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 a dilution series of an MMAE-conjugated antibody in 10% human serum in PBST in triplicate. Final concentration of the MMAE-conjugated antibody should cover the range from 0.01 ng/ml to 10,000 ng/ml. Include a zero MMAE-conjugated antibody concentration as the background value.
  7. Add 20 µl of an MMAE-conjugated 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 of HRP conjugated detection Anti-MMAE Antibody, TZA057 (AbD50348ad) 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 of QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.