Protocol: ADA Bridging ELISA for Use with Anti-Isatuximab Antibodies

Protocol: ADA Bridging ELISA for Use with Anti-Isatuximab Antibodies

Anti-Drug Antibody (ADA) Bridging ELISA: For Use with Anti-Isatuximab Monoclonal Antibodies Catalog HCA441, HCA442, HCA443

This method provides a procedure for generating an ADA ELISA standard curve with Anti-Isatuximab Antibody, HCA441, HCA442, HCA443, using isatuximab antibody for capture and detection. 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
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)
LYNX Rapid HRP Antibody Conjugation Kit™ (LNK001P-LNK006P)

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 detection antibody: conjugate isatuximab antibody using a LYNX Rapid HRP Antibody Conjugation Kit.
  2. Prepare the unconjugated isatuximab 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 isatuximab, 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 the Anti-Isatuximab Antibody, HCA441, HCA442, HCA443, in 10% human serum in PBST in triplicate. Final concentration of anti-isatuximab antibody should cover the range from 0.008 ng/mL to 32,000 ng/mL. Include a zero anti-isatuximab antibody concentration as the background value.
  7. Add 20 µL of anti-isatuximab antibody dilution per well (in triplicate for each standard recommended) and incubate for 1 hr at RT.
  8. Wash the microtiter plate 5x with PBST.
  9. To each well add 20 µL HRP-conjugated isatuximab diluted to 2 µg/mL in HISPEC Assay Diluent and 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.