The library was screened
against biotin-SBA and biotin-VVA using an enzyme-linked lectin assay
(ELLA), as described previously.52 (link),53 (link) A high-binding
96-well enzyme-linked immunosorbent assay (ELISA) plates (Greiner
Bio-One) was coated with the glycopeptide sublibraries and controls
(50 μL per well) at four different concentrations (0.1, 0.5,
10, and 500 μg/mL) in phosphate-buffered saline (PBS) (0.01
M, pH 7.4). Wells were incubated overnight to dry at 37 °C, and
then blocked with 3% bovine serum albumin (BSA) in PBS (300 μL)
overnight on the shaker. The coated wells were incubated with either
biotin-conjugated SBA or VVA lectin (100 μL, 50 μg/mL
in PBS) for 2 h at room temperature on the shaker. Upon washing with
PBS (2×, 100 μL), wells were treated with horseradish peroxidase
(HRP) conjugated streptavidin (100 μL, 1:4000) for 1 h at room
temperature on the shaker. The wells were then washed again with PBS
(2×, 100 μL) and water (300 μL), upon which 3,3,5,5-tetramethylbenzidine
(TMB) was added (100 μL) and incubated at room temperature for
15 min on the shaker. The reaction was terminated using 2 M sulfuric
acid (100 μL). The absorbance readings were recorded at 450
nm using an ELISA plate reader (BioTek, EPOCH plate reader). The average
absorbance reading after background subtraction was plotted against
the glycopeptide library concentration.
against biotin-SBA and biotin-VVA using an enzyme-linked lectin assay
(ELLA), as described previously.52 (link),53 (link) A high-binding
96-well enzyme-linked immunosorbent assay (ELISA) plates (Greiner
Bio-One) was coated with the glycopeptide sublibraries and controls
(50 μL per well) at four different concentrations (0.1, 0.5,
10, and 500 μg/mL) in phosphate-buffered saline (PBS) (0.01
M, pH 7.4). Wells were incubated overnight to dry at 37 °C, and
then blocked with 3% bovine serum albumin (BSA) in PBS (300 μL)
overnight on the shaker. The coated wells were incubated with either
biotin-conjugated SBA or VVA lectin (100 μL, 50 μg/mL
in PBS) for 2 h at room temperature on the shaker. Upon washing with
PBS (2×, 100 μL), wells were treated with horseradish peroxidase
(HRP) conjugated streptavidin (100 μL, 1:4000) for 1 h at room
temperature on the shaker. The wells were then washed again with PBS
(2×, 100 μL) and water (300 μL), upon which 3,3,5,5-tetramethylbenzidine
(TMB) was added (100 μL) and incubated at room temperature for
15 min on the shaker. The reaction was terminated using 2 M sulfuric
acid (100 μL). The absorbance readings were recorded at 450
nm using an ELISA plate reader (BioTek, EPOCH plate reader). The average
absorbance reading after background subtraction was plotted against
the glycopeptide library concentration.
