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

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

Anti-Drug Antibody (ADA) Bridging ELISA: For Use with Anti-Elotuzumab Monoclonal Antibodies Catalog HCA438, HCA439, and HCA440

This method provides a procedure for generating an ADA ELISA standard curve with Anti-Elotuzumab Antibody HCA438, HCA439, or HCA440, using elotuzumab 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 (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)
QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific, 15169)
LYNX Rapid Plus Biotin (Type 1) Antibody Conjugation Kit (LNK263B)
Neutravidin-HRP (Thermo Fisher Scientific 31030)

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 biotinylated elotuzumab detection antibody using LYNX Rapid Plus Biotin (Type 1) Antibody Conjugation Kit.
  2. Prepare the unconjugated elotuzumab 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 elotuzumab, 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-Elotuzumab Antibody HCA438, HCA439, or HCA440 in 10% human serum in PBST in triplicate. Final concentration of anti-elotuzumab antibody should cover the range from 0.015 ng/mL to 64,000 ng/mL. Include a zero anti-elotuzumab antibody concentration as the background value.
  7. Add 20 µL of anti-elotuzumab 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 biotinylated elotuzumab diluted to 2 µg/mL in PBST and incubate for 1 hr at RT.
  10. Wash the microtiter plate 5x with PBST.
  11. Add 20 µL Neutravidin-HRP (Thermo Fisher Scientific 31030, diluted 1:8,000) in HISPEC Assay Diluent and incubate for 1 hr at RT
  12. Wash the microtiter plate 10x with PBST.
  13. Add 20 µL QuantaBlu Fluorogenic Peroxidase Substrate to each well and measure the fluorescence after 30 min.