Establishment of a Monoclonal Antibody PMab-231 for Tiger Podoplanin

Introduction

Podoplanin (PDPN), also known as a T1alpha, is a type I transmembrane sialoglycoprotein that is expressed in several normal cells, including type I alveolar cells.⁽¹⁾ Therefore, PDPN is extremely useful for distinguishing type I alveolar cells from type II alveolar cells in pathological diagnosis.⁽²⁾ We previously reported that C-type lectin-like receptor-2 (CLEC-2) is an endogenous receptor of PDPN.^(3,4) Importantly, the PDPN/CLEC-2 interaction facilitates embryonic blood/lymphatic vessel separation.⁽⁵⁾ PDPN/CLEC-2 interaction also plays an essential role in murine lung development.⁽²⁾

The expression of human PDPN (hPDPN) has been reported in several malignant tumors, including brain tumors,⁽⁶⁾ oral squamous cell carcinomas,⁽⁷⁾ lung cancers,⁽⁸⁾ esophageal cancers,⁽⁹⁾ malignant mesotheliomas,⁽¹⁰⁾ osteosarcomas,⁽¹¹⁾ chondrosarcomas,⁽¹²⁾ and testicular tumors.⁽¹³⁾ The hPDPN expression is associated with cancer metastasis and malignant progression.^(4,6,14) Till date, we have developed monoclonal antibodies (mAbs) against human,⁽¹⁵⁾ mouse,⁽¹⁵⁾ rat,⁽¹⁶⁾ rabbit,⁽¹⁷⁾ bovine,⁽¹⁸⁾ dog,⁽¹⁹⁾ cat,⁽²⁰⁾ pig,⁽²¹⁾ and horse⁽²²⁾ PDPNs. However, anti-tiger PDPN mAbs have not been reported. In this study, we immunized mice with CHO/tigPDPN cells and established hybridomas that could produce mAbs against tigPDPN.

Materials and Methods

Results and Discussion

In this study, we employed the CBIS method to develop sensitive and specific mAbs against tigPDPN to facilitate the immunohistochemical analysis of paraffin-embedded tissue sections. Two mice were immunized with CHO/tigPDPN cells by following a screening procedure (Fig. 1). The developed hybridomas were seeded into 96-well plates and cultivated for 7 days. Wells positive for CHO/tigPDPN and negative for CHO-K1 were selected using flow cytometry. Screening identified strong signals against CHO/tigPDPN cells and weak or no signals against CHO-K1 cells in 18 of 958 wells (1.9%). One of the six clones, PMab-231 (IgG_2a, kappa), was finally selected using immunohistochemistry against paraffin-embedded feline normal tissue sections because tigPDPN and cPDPN shared 99% homology.⁽²³⁾

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FIG. 1.

Schematic illustration of CBIS method. Stable transfectants expressing tigPDPN used as an immunogen with no purification procedure. The selection of hybridomas secreting specific mAbs performed by flow cytometry using parental and transfectant cells. CBIS, Cell-Based Immunization and Screening; mAbs, monoclonal antibodies; tigPDPN, tiger podoplanin.

PMab-231 recognized CHO/tigPDPN, but showed no reaction with CHO-K1, as assessed using flow cytometry (Fig. 2). LpMab-17, an anti-LP tag mAb, detected CHO/tigPDPN. PMab-231 cross-reacted with cPDPN and horPDPN, but did not react with human, mouse, rat, rabbit, dog, bovine, pig, alpaca, bear, goat, sheep, Tasmanian devil, or whale PDPNs (Fig. 3).

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FIG. 2.

Detection of tigPDPN by flow cytometry using PMab-231. CHO/tigPDPN and CHO-K1 cells were treated with PMab-231 (red line) or anti-LP tag (LpMab-17; blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies. BSA, bovine serum albumin; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.

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FIG. 3.

Cross-reaction of PMab-231 to PDPNs of the other species using flow cytometry. CHO-K1 cells transfected with PDPNs of the other species were treated with PMab-231 (red line) or each positive control (blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies.

A kinetic analysis performed using flow cytometry assessed the interaction of PMab-231 with CHO/tigPDPN and CHO/cPDPN cells. K_D of PMab-231 for CHO/tigPDPN and CHO/cPDPN cells were determined to be 1.2 × 10⁻⁸ and 1.9 × 10⁻⁸, respectively, indicating moderate affinity for CHO/tigPDPN and CHO/cPDPN cells.

Western blotting performed using PMab-231 demonstrated that PMab-231 detected broad bands of tigPDPN and cPDPN between 35 and 48 kDa in CHO/tigPDPN and CHO/cPDPN cells, respectively (Fig. 4). LpMab-17, an anti-LP tag mAb and PMab-1, an anti-MAP tag mAb, detected each band in CHO/tigPDPN and CHO/cPDPN cells, respectively.^(24,32)

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FIG. 4.

Western blotting. Cell lysates of CHO-K1, CHO/tigPDPN, and CHO/cPDPN (10 μg) electrophoresed and transferred onto PVDF membranes. The membranes incubated with l μg/mL PMab-231, 10 μg/mL anti-LP tag (LpMab-17), 1 μg/mL anti-MAP tag (PMab-1), or 1 μg/mL anti-β-actin, and subsequently, with peroxidase-conjugated anti-mouse or -rat IgG. cPDPN, cat PDPN; PVDF, polyvinylidene difluoride.

The immunohistochemical analyses revealed that PMab-231 stained the feline lungs (Fig. 5) and kidneys (Fig. 6), but PMab-231 did not react with lymphatic endothelial cells (data not shown). These results indicate that PMab-231 is useful for the detection of cPDPN using immunohistochemistry. Because tigPDPN and cPDPN shared 99% homology, PMab-231 may be useful for the detection of tigPDPN using immunohistochemistry.

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FIG. 5.

Immunohistochemical analyses against the feline lungs. Tissue sections of the feline lungs incubated with 5 μg/mL PMab-231 (A, B) or with a blocking buffer (C, D), followed by the Envision+ kit. (E, F) Hematoxylin and eosin staining. Scale bar = 100 μm.

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FIG. 6.

Immunohistochemical analyses against the feline kidneys. Tissue sections of the feline kidneys incubated with 5 μg/mL PMab-231 (A, B) or with blocking buffer (C, D), followed by the Envision+ kit. (E, F) Hematoxylin and eosin staining. Scale bar = 100 μm.

In conclusion, we established a mAb against tigPDPN, PMab-231, which is suitable for use in flow cytometry, western blotting, and immunohistochemical analyses. The epitope of PMab-231 is required to be further investigated to clarify the sensitivity and specificity of PMab-231 against tigPDPN. It is expected that PMab-231 would prove useful for the elucidation of the pathophysiological functions of tigPDPN in future studies.