The anti-CD40 antibody concentration in the CAR-T cell supernatant was determined by ELISA. A total of 1 × 106 CAR-T cells were seeded in six-well plates coated with the rhMSLN antigen, and the supernatant of CAR-T cells was then collected after 24 h or 72 h.

A 96-well ELISA microplate was coated with 1 μg/ml rhCD40 (ACROBiosystems) at 4 °C overnight. After the microplate was sealed with 1% BSA for 2 h at 37 °C, the collected supernatant was added to the well and incubated for 1 h at 37 °C. After washing the wells three times, mouse anti-human IgG4 (HRP) (Abcam) was added to the well at a dilution of 1:30,000 and incubated for 30 min at 37 °C. After washing the wells three times, 3,3′,5,5′-tetramethylbenzidine (TMB) was added to develop the color. All of the experiments were performed in triplicate.

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