ELISA
was used for the analysis of induced anti-Muc1 antibodies in antiserum.
96-well MaxiSorp plates were coated by incubating overnight at 4 °C
with 1 μg/mL glycopeptide 11 in BSA, washed three times with
washing buffer (PBS containing 0.05% Tween-20), and blocked for 30
min at 37 °C with blocking buffer (washing buffer supplemented
with 1% BSA). Dilutions of sera in blocking buffer were added and
incubated for 2 h at 37 °C. The plates were washed three times
with washing buffer and incubated with the different antibody subtypes
IgG1, IgG2b (BD Pharmigen), IgG2a (PharMingen), IgG2c (Abcam), and
IgG3 (BioLegend). After incubation and three washes, horseradish peroxidase-conjugated
goat anti-mouse IgG (H + L) (Abcam) diluted 1:1000 in blocking buffer
was added for 1 h at 37 °C. The plates were then washed three
times and incubated for 10 min with 50 μL per well of 2,2′-azino-di-(3-ethylbenzothiazoline
sulfonic acid substrate in citrate buffer (pH 4.4 and 1:4000) 30%
H2O2. The reaction was quenched by adding 50
μL of 1 M aqueous sulfuric acid to each well. Plates were read
at 410 nm using a Tecan Infinite F200 Pro microplate reader. For each
IgG of the antiserum, optical densities were measured and the absorbance
curves were fitted to obtain antibody titers. There were no IgGs detectable
for the negative controls (untreated mice).
was used for the analysis of induced anti-Muc1 antibodies in antiserum.
96-well MaxiSorp plates were coated by incubating overnight at 4 °C
with 1 μg/mL glycopeptide 11 in BSA, washed three times with
washing buffer (PBS containing 0.05% Tween-20), and blocked for 30
min at 37 °C with blocking buffer (washing buffer supplemented
with 1% BSA). Dilutions of sera in blocking buffer were added and
incubated for 2 h at 37 °C. The plates were washed three times
with washing buffer and incubated with the different antibody subtypes
IgG1, IgG2b (BD Pharmigen), IgG2a (PharMingen), IgG2c (Abcam), and
IgG3 (BioLegend). After incubation and three washes, horseradish peroxidase-conjugated
goat anti-mouse IgG (H + L) (Abcam) diluted 1:1000 in blocking buffer
was added for 1 h at 37 °C. The plates were then washed three
times and incubated for 10 min with 50 μL per well of 2,2′-azino-di-(3-ethylbenzothiazoline
sulfonic acid substrate in citrate buffer (pH 4.4 and 1:4000) 30%
H2O2. The reaction was quenched by adding 50
μL of 1 M aqueous sulfuric acid to each well. Plates were read
at 410 nm using a Tecan Infinite F200 Pro microplate reader. For each
IgG of the antiserum, optical densities were measured and the absorbance
curves were fitted to obtain antibody titers. There were no IgGs detectable
for the negative controls (untreated mice).