Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.


Hybridoma production
Female BALB/c mice (6 weeks old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogenfree conditions. The Animal Care and Use Committee of Tohoku University approved all the animal experiments. Two BALB/c mice were immunized with CHO/aPDPN cells (1 × 10 8 ) intraperitoneally (i.p.) administered together with Imject Alum (Thermo Fisher Scientific Inc.). The procedure included three additional immunizations, followed by a final booster injection administered i.p. two days prior to the harvest of spleen cells, amounting to a total of five immunizations. These spleen cells were subsequently fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), and the hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). The cultured supernatants were screened using flow cytometry.

Determination of binding affinity using flow cytometry
CHO/aPDPN was suspended in 100 μL of serially diluted PMab-225, followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.). Fluorescence data were collected using EC800 Cell Analyzer (Sony Corp.). The dissociation constant (K D ) was obtained by fitting the binding isotherms to built-in onesite binding models in GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA, USA).

Immunohistochemical analyses
Normal alpaca tissues were collected after autopsy at Hokkaido University, fixed in 10% neutral-buffered formalin [36], and routinely processed to make paraffin-embedded tissue sections. Histological sections of 4 μm thickness were directly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies Inc.) for 20 min. These tissue sections were blocked using SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), incubated with PMab-225 (1 μg/mL or 5 μg/mL) for 1 h at room temperature, and treated using an Envision + Kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Results
In this study, two mice were immunized with CHO/aPDPN cells (Fig. 1). Developed hybridomas were seeded into 96-well plates and cultivated for 8 days (first mouse) or 9 days (second mouse). Wells positive for CHO/aPDPN and negative for CHO-K1 were selected using flow cytometry. Screening identified strong signals against CHO/ aPDPN cells and weak or no signals against CHO-K1 cells in 83 of 960 wells (8.6%). Of these 83 wells, two hybridomas were developed. One of these two clones, PMab-225 (IgG 2b , kappa), was selected for immunohistochemistry against alpaca tissues.
The immunohistochemical analyses using antigen retrieval with citrate buffer (pH 6.0) revealed that PMab-225 strongly stained type I alveolar cells in the alpaca lung (Fig. 4) and lymphatic endothelial cells in alpaca colon tissues (Fig. 5). Podocytes and Bowman's capsule of alpaca kidney were stained using antigen retrieval with EnVision FLEX Target Retrieval Solution High pH (Fig. 6). These results indicate that PMab-225 will be useful to elucidate the pathophysiological functions of aPDPN in alpaca tissues in the future.

Discussion
In our previous studies, we established a cancer-specific monoclonal antibody (CasMab) technology to produce CasMabs, such as LpMab-2 and LpMab-23 against hPDPN, in several studies [17,37]. Those Cas-Mabs against hPDPN can detect only hPDPN-expressing cancer cells, not normal cells, including lymphatic endothelial cells and pulmonary type I alveolar cells. Although LpMab-2 might bind to both a peptide and glycans of hPDPN [17], LpMab-23 could detect the conformational change of hPDPN peptides, which might be induced by cancer-specific glycans [38]. Both LpMab-2 and LpMab-23 possess high antitumor activities by those antibody-dependent cellular cytotoxicities (ADCC) [38,39]. Furthermore, LpMab-23-recognizing cancer-type podoplanin could be a novel predictor for a poor prognosis of early stage tongue cancer [40]. Recently, we also utilized a Cell-Based Immunization and Screening (CBIS) method to establish mAbs against various membrane proteins, such as CD133 [41], CD44 [42], PD-L1 [43], pig PDPN [34], horse PDPN [44], and cat PDPN [32]. Importantly, those mAbs are very useful for flow cytometry, Western blot, and immunohistochemistry. In contrast, we could not develop useful mAbs by immunizing synthetic peptides (data not shown). Using selecting one method or the combination of those methods such as CasMab technology and CIBS method, we could produce sensitive and specific mAbs against membrane proteins, which are very useful for not only flow cytometry, but also Western blot and immunohistochemistry when we could not develop mAbs by immunizing synthetic peptides or recombinant proteins. Indeed, we first tried to produce anti-aPDPN mAbs by immunizing synthetic peptides, which are corresponding to PLAG domains of aPDPN; however, we could not obtain any mAbs, which are applicable for Western blot or immunohistochemistry (data not shown). Then, we employed the CBIS method in this study to develop sensitive and specific mAbs against aPDPN for the immunohistochemical analysis of paraffin-embedded tissue sections (Fig. 1). Finally, PMab-225, which is very useful for flow cytometry (Fig. 2), Western blot (Fig. 3), and immunohistochemical analyses (Figs. 4-6), was developed. Interestingly, PMab-225 cross-reacted with human, bovine, tiger, bear, goat, sheep, and whale PDPNs, which were overexpressed in CHO-K1 cells (data not shown), although the percentage of homology of aPDPN with hPDPN is only 66%. In contrast, PMab-225 did not react with mouse, rat, rabbit, dog, cat, pig, Tasmanian devil, and horse PDPNs (data not shown). In future study, we should determine the critical epitope of PMab-225; then, we might uncover the mechanism of cross-reactivity against many species. In immunohistochemical analysis, PMab-225 stained lymphatic endothelial cells (Fig. 5) and pulmonary type I alveolar cells using antigen retrieval with citrate buffer (Fig. 4). However, PMab-225 did not stain alpaca kidney in this condition (data not shown). In contrast, alpaca kidney was stained using antigen retrieval with EnVision FLEX Target Retrieval Solution High pH (Fig. 6). In the future study, we should clarify the molecular difference of aPDPNs, including posttranslational modifications in several tissues.
In conclusion, we have established a mAb against aPDPN, PMab-225, which is suitable for use in flow cytometry, Western blotting, and immunohistochemical analyses. PMab-225 should prove useful to elucidate the pathophysiological functions of aPDPN in future studies. In contrast, sensitive and specific mAbs against membrane proteins for alpaca have not been established; therefore, we should develop many mAbs against alpaca membrane proteins, such as CD31 or LYVE-1 for investigation of vascular endothelial cells or lymphatic endothelial cells.

Conflicts of interest
The authors declare no conflicts of interest involving this article.