Specific Detection of Dog Podoplanin Expressed in Renal Glomerulus by a Novel Monoclonal Antibody PMab-38 in Immunohistochemistry

Cell lines, dog tissues, and animals

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). CHO-K1, stable CHO transfectants, and P3U1 were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Female BALB/c mice (4 weeks old) were purchased from CLEA Japan (Tokyo, Japan). Normal dog tissues were obtained from North lab (Hokkaido, Japan). Animals were housed under pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.

Hybridoma production

The dPDPN with N-terminal PA tag and C-terminal RAP tag-MAP tag (PA-dPDPN-RAP-MAP) was inserted into pCAG-zeo vector (Wako Pure Chemical Industries Ltd., Osaka, Japan). RAP tag consists of 12 amino acids (DMVNPGLEDRIE) and MAP tag consists of 12 amino acids (GDGMVPPGIEDK). CHO-K1 was transfected with pCAG-zeo/PA-dPDPN-RAP-MAP using Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA). For the purification of PA-dPDPN-RAP-MAP from cell membrane, we used the PA tag system.^(28,29) BALB/c mice were immunized by intraperitoneal (i.p.) injection of 100 μg of recombinant PA-dPDPN-RAP-MAP together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations of 50 μg, a booster injection of 100 μg was given i.p. 2 days before spleen cells were harvested. The spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).

The culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to purified recombinant PA-dPDPN-RAP-MAP. Recombinant proteins of PA-dPDPN-RAP-MAP were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 1 μg/mL for 30 minutes. 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 1:3000 diluted peroxidase-conjugated antimouse IgG (Dako; Agilent Technologies, Inc., Santa Clara, CA). 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.).

Immunohistochemical analyses

Four-micrometer-thick histologic sections were deparaffinized in xylene and rehydrated and were autoclaved in citrate buffer (pH 6.0; Dako) for 20 minutes. Sections were incubated with 10 μg/mL of primary mAbs for 1 hour at room temperature followed by treatment with Envision+ kit for 30 minutes (Dako). Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Dako) for 1 minute, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.).

Flow cytometry

Stable transfectant of CHO/PA-dPDPN-RAP-MAP was established using SH800 (Sony Corp., Tokyo, Japan). The other stable transfectants [CHO/PA-bovine PDPN (bovPDPN)-RAP-MAP, CHO/hPDPN-FLAG, CHO/mouse PDPN (mPDPN)-FLAG, CHO/rat PDPN (rPDPN)-His, and CHO/PA-rabbit PDPN (rabPDPN)] were previously established.^(1,30–32) Cells were harvested by brief exposure to 0.25% trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washing with 0.1% BSA/PBS, the cells were treated with primary mAbs (1 μg/mL) for 30 minutes at 4°C followed by treatment with Oregon green-conjugated antimouse IgG (1:1000 diluted; Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp.).

Western blot analysis

Cell lysates (10 μg) of CHO/PA-dPDPN-RAP-MAP, CHO/bovPDPN-FLAG, CHO/hPDPN-FLAG, CHO/mPDPN-FLAG, CHO/rPDPN-His, CHO/PA-rabPDPN, and CHO-K1 were boiled in SDS sample buffer (Nacalai Tesque, Inc.). The proteins were electrophoresed on 5%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd.) and were transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore Corp., Billerica, MA). After blocking with 4% skim milk (Nacalai Tesque, Inc.), the membrane was incubated with PMab-38 and anti-β-actin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO) and then with peroxidase-conjugated antimouse IgG (1:1000 diluted; Dako) and developed with the Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific, Inc.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Production of mAbs against dPDPN

We immunized mice with the recombinant proteins, which were purified from CHO/PA-dPDPN-RAP-MAP cells, and the ELISA screening was performed. Anti-PA tag mAb (clone: NZ-1), anti-MAP tag mAb (clone: PMab-1), and anti-RAP tag mAb (clone: PMab-2) detected the PA-dPDPN-RAP-MAP protein in Western blot analysis (data not shown). Among ELISA-positive wells, the second screening was performed by flow cytometry. Finally, a positive wells were selected in immunohistochemical analysis. After limiting dilution, one of the clones, PMab-38 (IgG₁, kappa), was established. PMab-38 reacted with podocytes of renal glomerulus (Fig. 1A), whereas it did not react with other normal cells such as LECs (Fig. 1B) or type I alveolar cells of lung (Fig. 1C), in which PDPN expression has been reported in other species.^(3,7) These data indicate that PMab-38 is useful for immunohistochemistry using paraffin-embedded tissues.

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FIG. 1.

Immunohistochemical analysis by PMab-38. Sections of dog kidney (A), dog colon (B), and dog lung (C) were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 10 μg/mL of PMab-38, followed by EnVision+ kit, and color was developed using DAB and counterstained with hematoxylin. PBS was used as negative control. HE staining was performed against serial sections. Arrow head, renal glomerulus. Scale bar: 100 μm. HE, hematoxylin and eosin.

Specificity of PMab-38 against dPDPN in flow cytometry and Western blot

PMab-38 reacted with CHO/PA-dPDPN-RAP-MAP, not with CHO/PA-bovPDPN-RAP-MAP, CHO/hPDPN-FLAG, CHO/mPDPN-FLAG, CHO/rPDPN-His, CHO/PA-rabPDPN, and CHO-K1 cells in flow cytometry (Fig. 2A). In Western blot analysis, PMab-38 recognized only dPDPN of CHO/PA-dPDPN-RAP-MAP, and did not react with PDPNs of CHO/bovPDPN-FLAG, CHO/hPDPN-FLAG, CHO/mPDPN-FLAG, CHO/rPDPN-His, CHO/PA-rabPDPN, or CHO-K1 cells (Fig. 2B). These results indicate that PMab-38 detects dPDPN specifically.

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FIG. 2.

Flow cytometric and Western blot analyses by PMab-38. (A) Cells were treated with PMab-38 followed by treatment with Oregon green-conjugated antimouse IgG. Black line: negative control. (B) Cell lysates (10 μg) were electrophoresed and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of mAbs (PMab-38 or anti-β-actin), and then with peroxidase-conjugated antimouse IgG; the membrane was developed with Pierce Western Blotting Substrate Plus and detected using a Sayaca-Imager. mAbs, monoclonal antibodies; PVDF, polyvinylidene difluoride.