Lightning link streptavidin conjugation kit

The design of the IL-1α secretion assay was adapted based on a previous report^{50 (link)}. T_H17 cells (1 × 10⁶) were stained with 1 mg ml⁻¹ of sulfo-NHS-LC-biotin (catalog no. ab145611, Abcam), incubated for 30 min at room temperature and then washed 3× with PBS (pH 8) supplemented with 100 mM glycine. The final washing of cells was performed with PBS supplemented with 0.5% bovine serum albumin. Cell surface biotinylation was validated with PE-labeled streptavidin (catalog no. 554061, BD Pharmingen). Purified anti-human IL-1α antibodies (AF-200-NA, R&D) were labeled with streptavidin using a Lightning-Link Streptavidin Conjugation kit (catalog no. ab102921, Abcam). For cytokine secretion, cells were stimulated with anti-CD3 and anti-CD28 for 72 h. The cells were collected and labeled with streptavidin-IL-1α and incubated for 24 h on the MACSmix tube rotator (Miltenyi Biotec). Recombinant IL-1α (Miltenyi Biotec) was added as a positive control. The cells were then stained with a PE-labeled IL-1α antibody (clone 364-3B3-14, BioLegend, 1:50).

The CD47 antibody was conjugated to streptavidin for 3 h using the Lightning‐Link Streptavidin Conjugation Kit (Abcam, ab102921) according to the manufacturer's protocol. Mertansine (DM1) was biotinylated at a molar ratio of 8:1 using EZ‐Link BMCC‐biotin according to the manufacturer's instructions. Biotinylated DM1 was added to CD47 antibody–streptavidin at a ratio of 3.5:1 to match a clinically available anti‐CD47‐DM1 drug‐to‐antibody ratio (DAR) of 3.5, and the mixture was incubated at room temperature for 30 min. Anti‐CD47‐DM1 was purified using a NAb Protein G spin column (89953, Thermo Scientific) and resuspended in PBS for functional experiments. The purity and drug–antibody ratio (DAR) was analyzed using NanoDrop 2000 (Thermo Fisher Scientific, USA), and DAR was calculated following the published method:^[44] (ε_mAb²⁵²‐Rε_mAb²⁸⁰)/(Rε_Drug²⁸⁰‐ε_Drug²⁵²), where R = A₂₅₂/A₂₈₀ = absorbance ratio, ε_mAb²⁵² = 9.41 ×10⁴ M⁻¹ cm⁻¹, ε_mAb²⁸⁰ = 2.34 × 10⁵ M⁻¹ cm⁻¹, ε_DM1²⁵² = 2.64 × 10⁵ M⁻¹ cm⁻¹, ε_DM1²⁸⁰ = 5.23 × 10³ M⁻¹ cm⁻¹. The integrity of ADC was confirmed with SDS‐PAGE.