MUC18 protein
Enzyme-linked immunosorbent assay (ELISA) is one of the most specific and straightforward assays for detecting biomolecules in research and clinics. ELISA was used to analyze whether the ability of the mAb to bind to the MUC18 target changed before and after DFO conjugation. First, the MUC18 protein (Sino Biological) was diluted to 1 µg/mL with coating solution (0.1 M carbonate buffer, pH 9.5) and then plated on a 96-microtiter plate well (100 µL/well) with a pipette at 4 °C overnight. After the 96-well plates were washed with PBST 5 times, nonspecific sites were blocked with 5% skim milk powder (200 µL/well, 37 °C, 2 h), and the plates were subsequently discarded and washed with PBST 5 times. The concentrations of the primary antibodies IP150 and DFO-IP150 were diluted to 0.000032-9.9 µg/mL, and then 100 µL was added to each well (n=4, 37 °C, 2 h). Then, the cells were incubated with a rabbit anti-human secondary antibody (1:3000, 100 µL/well, 37 °C, 2 h). After incubation with TMB (100 µL/well, Solarbio) in the dark for 3-5 minutes, 12.5% H2SO4 (50 µL/well) was added to terminate the color development, and the OD450 nm was read with an enzyme marker.
2.5 Micro-PET/CT Imaging in Melanoma Tumor Xenografts
Xenograft tumor models (CDX/PDX) were used to noninvasively evaluate the specificity and targeting of the 89Zr-labeled antibody against MUC18 in vivo. Experiments were performed when the tumors reached 5–15 mm in diameter. Images of models injected with 3.7 MBq89Zr-IP150 (200 µL, 100 µCi) via the tail vein were collected at specific time points (4, 24, 48, 72, 96, 120 and 168 h, p.i.). One milligram of IP150 was injected intravenously in the blocked group in advance.
The PET collection time was 900 s, and CT images were acquired after PET collection and reconstruction. Images were processed using VivoQuant postprocessing software. PET imaging raw data were converted into false-colored maximum intensity projections on a color scale expressed as SUVs, and quantitative results were obtained by mapping regions of interest (ROIs).