Evaluating mCAR-MAGE-A1 Binding via ELISA

A sandwich ELISA was performed to evaluate the binding ability of mCAR to MAGE-A1 as described before [34 (link)]. Briefly, 96-well plates were seeded with transfected 293T cells (mCAR and unrelated-CAR). Untransfected 293T cells (blank) were used as a negative control. Then, each well was washed and MAGE-A1 antigens were added (Novus Biologicals, Littleton, CO, USA) at different dilutions. Then, the supernatants were collected and added to another 96-well plate, which was preliminarily coated with anti-MAGE-A1 rabbit polyclonal antibody (LS-C327797-200, LifeSpan BioSciences, Seattle, WA, USA), followed by the addition of a primary anti-MAGE-A1 mouse monoclonal antibody (LS-C25368-100, LifeSpan BioSciences) and a secondary anti-mouse antibody. After washing, the optical density at 450 nm (OD450) was measured with an automatic microplate reader (Thermo Fisher Scientific, USA). The supernatant lentivirus titers were detected following the protocol described previously [35 (link), 36 (link)].