Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.


Hybridoma production
We employed a Cell-Based Immunization and Screening (CBIS) method [25,33,35,36] to develop sensitive and specific mAbs against tasPDPN. Briefly, two BALB/c mice were immunized with CHO/ tasPDPN cells (1 × 10 8 ) 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 ip. 2 days prior to the harvest of spleen cells. Subsequently, these spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), 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 following a brief exposure to 0.25% trypsin and 1 mM ethylendiaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and treated with primary mAbs for 30 min at 4°C. Thereafter, the cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo Fisher Scientific Inc.). Then, fluorescence data were collected using the SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).   collected using the EC800 Cell Analyzer (Sony Corp.). 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, USA).

Results and discussion
Most cancers are somatic in origin, and only a few transmissible cancers have been documented [37]. Transmissible cancers have been reported only in natural cases, such as canine transmissible venereal tumor in dogs [38] or devil facial tumor disease in Tasmanian devils [39]. Tasmanian devils (Sarcophilus harrisii) are endangered owing to the emergence of two clonally transmissible cancers: devil facial tumor disease 1 (DFT1) and devil facial tumor disease 2 (DFT2). DFT1 and DFT2 are infectious diseases that spread via biting [40]. DFT1 was first discovered in northeastern Tasmania in 1996 and has since then spread to more than 80% of the area across the island, causing a significant decrease in the population [41]. DFT2 was discovered in 2014 and is currently restricted to a small region of southeastern Tasmania [42]. Although we had previously developed mAbs against human [20], mouse [20], rat [21], rabbit [22], bovine [23], dog [24], cat [25], pig [43], and horse [27] PDPNs, mAbs against tasPDPN has not yet been developed. The development of anti-tasPDPN mAbs will enable us to perform pathophysiological studies about the lymphatic metastasis or lymphangiogenesis.
In the present study, we employed the CBIS method to develop sensitive and specific mAbs against tasPDPN to facilitate the immunohistochemical analysis of paraffin-embedded tissue sections. Two mice were immunized with CHO/tasPDPN cells using an immunization and screening procedure (Fig. 1). The developed hybridomas were seeded into 96-well plates and cultivated for 9 days. Wells positive for CHO/tasPDPN and negative for CHO-K1 were selected by flow cytometry. The screening approach identified strong signals from CHO/ tasPDPN cells and weak or no signals from CHO-K1 cells in 19 of the 960 wells (2.0%). After limiting dilution of 19 wells, we developed nine clones. One of these nine clones, PMab-233 (IgG 1 , kappa), was finally selected via immunohistochemistry against the paraffin-embedded sections of CHO/tasPDPN cell.
In conclusion, we established an mAb, PMab-233, against tasPDPN, which is suitable for use in flow cytometry, Western blotting, and immunohistochemical analyses. The epitope of PMab-233 needs further investigation to clarify the sensitivity and specificity of PMab-233 against tasPDPN. We believe that PMab-233 should prove to be useful in elucidating the pathophysiological functions of tasPDPN in future studies.

Conflicts of interest
Y.K. received research funding from ZENOAQ RESOURCE CO., LTD. The other authors have no conflict of interest.