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).