Nunc maxisorp 96 well immunoplates

Synthesized ATRX peptides using PEPScreen (Sigma-Aldrich Corp., St. Louis, MO, USA) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 10 μg/mL for 30 min at 37 °C. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the plates were incubated with 10 μg/mL purified AMab-6 followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). These reactions were performed at 37 °C using a total sample volume of 50–100 μL.

The mCCR9 peptides, such as wild type (WT), 19 of 1× alanine (1× Ala)-substituted peptides (Table 1), and 18 of 2× alanine (2× Ala)-substituted peptides (Table 2), were synthesized using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO, USA). Each peptide was immobilized on Nunc Maxisorp 96-well immuno plates (Thermo Fisher Scientific, Inc.) at a concentration of 1 μg/mL for 30 min at 37 °C. As a negative control, no peptide was immobilized on the immuno plates. After washing with phosphate-buffered saline containing 0.05% Tween20 (PBST), the wells were blocked with 1% bovine serum albumin containing PBST for 30 min at 37 °C. The plates were then incubated with C₉Mab-24 (1 μg/mL), followed by a 1:20,000 dilution of peroxidase-conjugated anti-rat immunoglobulins (Sigma-Aldrich Corp.). Enzymatic reactions were performed using an ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was detected at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).

Recombinant hPDPN or glycopeptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at a concentration of 1 μg/ml for 30 min. After blocking with 1% BSA in 0.05% Tween20/phosphate buffered saline (PBS, Nacalai Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG or anti-rat IgG (Dako; Agilent Technologies, Inc., Glostrup, Denmark). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories Inc.).

Fifty-eight peptides, which cover the extracellular domain of CD44v3–10 [26 (link)], were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). We immobilized them on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc) at 1 µg/mL for 30 min at 37 °C. The palate washing was performed using the HydroSpeed Microplate Washer (Tecan, Zürich, Switzerland) with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After the blocking with 1% (w/v) bovine serum albumin (BSA) in PBST for 30 min at 37 °C, C₄₄Mab-1 (10 µg/mL) was added to each well. Then, the wells were further incubated with anti-mouse immunoglobulins peroxidase-conjugate (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA) for 30 min at 37 °C. One-Step Ultra TMB (Thermo Fisher Scientific Inc.) was used for enzymatic reactions. An iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) was used to measure the optical density at 655 nm.

Fifty-eight synthesized peptides (Sigma-Aldrich Corp., St. Louis, MO, USA), which cover the CD44v3-10 extracellular domain [21 (link)], were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc) at a concentration of 1 µg/mL for 30 min at 37 °C. After washing with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.) using Microplate Washer, HydroSpeed (Tecan, Zürich, Switzerland), wells were blocked with 1% (w/v) bovine serum albumin (BSA)-containing PBST for 30 min at 37 °C. C₄₄Mab-9 was added to each well, and then incubated with peroxidase-conjugated anti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA). Enzymatic reactions were performed using 1 Step Ultra TMB (Thermo Fisher Scientific Inc.). The optical density at 655 nm was measured using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).

Synthesized rPDPN peptides using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO) were immobilized on Nunc MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 10 μg/mL for 30 minutes at 37°C. After blocking with Superblock T20 (PBS) blocking buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified PMab-2 (10 μg/mL), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc.). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark Microplate Reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37°C with a total sample volume of 50–100 μL.

Synthesized EGFR (Accession No.: NP_005219) peptides using PEPScreen (Sigma-Aldrich Corp., St. Louis, MO) and extracellular domain of EGFR (EGFRec) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at 10 μg/ml for 30 min at 37 °C or over night at 4 °C. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the plates were incubated with purified EMab-134 (10 μg/ml), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc., Santa Clara, CA). The enzymatic reaction was conducted using 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37 °C with a total sample volume of 50–100 μl.