Establishment of an Anticetacean Podoplanin Monoclonal Antibody PMab-237 for Immunohistochemical Analysis

Cell lines

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding wPDPN (accession no.: XM_007104824.2) plus an N-terminal RIEDL tag, which are recognized by an anti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Plasmids were transfected using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were selected by limiting dilution and cultivation in a medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto, Japan).

The P3U1, CHO-K1, CHO/wPDPN, CHO/hPDPN,⁽²⁴⁾ CHO/mouse PDPN (mPDPN),⁽²⁴⁾ CHO/rat PDPN (rPDPN),⁽¹³⁾ CHO/rabbit PDPN (rabPDPN),⁽¹⁴⁾ CHO/dog PDPN (dPDPN),⁽¹⁶⁾ CHO/bovPDPN,⁽¹⁵⁾ CHO/cPDPN,⁽¹⁷⁾ CHO/pig PDPN (pPDPN),⁽²⁵⁾ CHO/horse PDPN (horPDPN),⁽²⁶⁾ CHO/tigPDPN,⁽²⁰⁾ CHO/alpaca PDPN (aPDPN),⁽²³⁾ CHO/bear PDPN (bPDPN),⁽²⁵⁾ CHO/Tasmanian devil PDPN (tasPDPN),⁽²⁵⁾ CHO/goat PDPN (gPDPN),⁽²¹⁾ and CHO/sheep PDPN (sPDPN)⁽²²⁾ were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), supplemented with 10% of heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). The cells were grown in an incubator at 37°C with humidity and 5% CO₂ and 95% air atmosphere.

Animals

Female BALB/c mice (6 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). The animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments.

Hybridoma production

We employed a Cell-Based Immunization and Screening (CBIS) method^(17,27–29) to develop mAbs against wPDPN. In brief, two BALB/c mice were immunized with CHO/wPDPN cells (1 × 10⁸) intraperitoneally (i.p.) together with the Imject Alum (Thermo Fisher Scientific, Inc.). The procedure included three additional immunizations, followed by a final booster injection administered i.p. 2 days before the harvest of spleen cells. Subsequently, these spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN), and the hybridomas were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific, Inc.). The culture supernatants were screened by flow cytometry.

Flow cytometry

The cells were harvested after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin in phosphate-buffered saline (PBS) and treated with primary mAbs for 30 minutes at 4°C. Thereafter, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA) or Oregon Green anti-rat IgG (1:2000; Thermo Fisher Scientific, Inc.). Then, fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corporation, Tokyo, Japan).

Determination of binding affinity by flow cytometry

CHO/wPDPN was suspended in 100 μL of serially diluted PMab-237. Then, Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were collected using the EC800 Cell Analyzer (Sony Corporation). The dissociation constant (K_D) was calculated by fitting the binding isotherms to built-in one-site binding models in the GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

Western blot analysis

Cell lysates (10 μg) were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). Proteins were then electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 1 μg/mL of PMab-237, 10 μg/mL of LpMab-7, and 1 μg/mL of anti-β-actin (clone AC-15; Sigma-Aldrich Corporation, 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 (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Immunohistochemical analyses

Cell blocks were produced using iPGell (Genostaff Co., Ltd., Tokyo, Japan). Histological sections of 4-μm thickness were deparaffinized in xylene, then rehydrated, and autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes. Then, sections were blocked using the SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), incubated with PMab-237 (1 μg/mL) for 1 hour at room temperature, and treated with the Envision+ Kit for mouse (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).