Peroxidase conjugated anti mouse igg

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.).

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.

Cell lysates (10 μg) were boiled in SDS sample buffer (Nacalai Tesque, Inc.). Proteins were then electrophoresed on 5%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with anti-EGFR (1–10 μg/mL) and 1 μg/mL of anti-β-actin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO), followed by incubation with peroxidase-conjugated anti-mouse IgG (diluted 1:1000; Agilent Technologies, Inc., Santa Clara, CA), and were finally developed using ImmunoStar LD (Wako Pure Chemical Industries Ltd.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

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.

Three 4‐week‐old female BALB/c mice were immunized by intraperitoneal (i.p.) injection of 1 × 10⁸ LN229/hPDPN cells together with Imject Alum (Thermo Fisher Scientific Inc.) 30. A booster injection was administered i.p. 2 days before the mice were euthanized by cervical dislocation. Spleen cells were harvested and fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were cultured in RPMI 1640 medium containing hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific Inc.). The culture supernatants were screened, using an enzyme‐linked immunosorbent assay (ELISA) and recombinant human PDPN purified from LN229/hPDPN cells 30. Proteins (1 μg/mL) were immobilized on Nunc Maxisorp 96‐well immunoplates (Thermo Fisher Scientific Inc.) for 30 min. After blocking with 1% bovine serum albumin (BSA) in 0.05% Tween20/phosphate‐buffered saline (PBS) (Nacalai Tesque, Inc.), the plates were incubated with culture supernatants, followed by the addition of peroxidase‐conjugated anti‐mouse IgG diluted 1:2000 (Dako; Agilent Technologies, Inc.). The enzymatic reaction was conducted using a 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.).

Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.) and proteins were then electrophoresed on 5%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 10 μg/mL of anti-EGFR (clone EMab-51) and 1 μg/mL of anti-β-actin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO), then with peroxidase-conjugated anti-mouse IgG (1:1000 diluted; Agilent Technologies, Inc., Santa Clara, CA), and were finally developed using ImmunoStar LD (Wako Pure Chemical Industries Ltd.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).