PMab-322: a novel anti-hippopotamus podoplanin monoclonal antibody for multiple applications

Abstract

Podoplanin (PDPN) is a highly glycosylated type I transmembrane protein. PDPN expression is observed in various normal tissues, including lymphatic endothelial cells, kidney podocytes, and type I alveolar epithelial cells in the lungs. Monoclonal antibodies (mAbs) targeting PDPN across different animal species have facilitated the identification of PDPN-positive cells. To date, we have developed anti-PDPN mAbs for over 20 species. These antibodies suit various applications, including flow cytometry, immunoblotting, and immunohistochemistry. In this study, we generated an anti-hippopotamus PDPN (hipPDPN) mAb, PMab-322 (mouse IgG_2a, kappa), using the Cell-Based Immunization and Screening (CBIS) method. PMab-322 exhibited strong reactivity to hipPDPN-overexpressed Chinese hamster ovary-K1 and demonstrated moderate affinity (K _D: 4.4 × 10^-8 M) in a flow cytometry-based measurement. PMab-322 specifically recognizes hipPDPN but does not cross-react with PDPN from 23 other species. Furthermore, PMab-322 successfully detected hipPDPN in both immunoblotting and immunohistochemistry. These findings highlight the potential of PMab-322 for pathological analyses of hippopotamus-derived tissues.

Keywords: CBIS method; Flow cytometry; Hippopotamus podoplanin; Immunoblotting; Immunohistochemistry; Monoclonal antibody.

Fig. 1

A schematic illustration of anti-hipPDPN mAbs production. (A) CHO/MAP16-hipPDPN was immunized into BALB/cAJcl mice. (B) The spleen cells were fused with P3U1 cells. (C) To select anti-hipPDPN mAb-producing hybridomas, the supernatants were screened by flow cytometry using CHO/PA16-hipPDPN and CHO–K1. (D) After limiting dilution, anti-hipPDPN mAbs were cloned by limiting dilution. PMab-322 (mouse IgG_2a, kappa) was finally established.

Fig. 2

Flow cytometric analysis of PMab-322 against CHO/PA16-hipPDPN. (A) CHO/PA16-hipPDPN and CHO–K1 were treated with 10–0.01 μg/mL of PMab-322 (red line) or blocking buffer (black line), followed by Alexa Fluor 488-conjugated anti-mouse IgG. (B) CHO/PA16-hipPDPN was suspended in 100 μL serially diluted PMab-322. Then, cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were subsequently collected using the SA3800 Cell Analyzer. The dissociation constant (K_D) of PMab-322 was determined by GraphPad PRISM 6.

Fig. 3

Specificity of PMab-322 against 24 species PDPN-overexpressed CHO–K1. (A) Twenty-four species PDPN-overexpressed CHO–K1 cell lines were treated with 10 μg/mL of PMab-322 (red line) or blocking buffer (black line), followed by Alexa Fluor 488-conjugated anti-mouse IgG. (B) The expression of each PDPN was confirmed by corresponding anti-PDPN mAbs (green line, 10 μg/mL). Then, Alexa Fluor 488-conjugated anti-mouse IgG or anti-rat IgG were treated. Fluorescence data were collected using the SA3800 Cell Analyzer. Note that the recognition of CHO/PA16-hipPDPN by NZ-1 was mediated by the reaction to PA16-tag.

Fig. 4

Detection of hipPDPN by immunoblotting. The membranes, on which cell lysates of CHO–K1 and CHO/PA16-hipPDPN were transferred, were incubated with 1 μg/mL of PMab-322, 1 μg/mL of NZ-1, or 1 μg/mL of RcMab-1. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse (for PMab-322) or horseradish peroxidase-conjugated anti-rat immunoglobulins (for NZ-1 and RcMab-1). Chemiluminescence signals were developed and detected with a Sayaca-Imager.

Fig. 5

Immunohistochemistry of paraffin-embedded sections of CHO/PA16-hipPDPN and CHO–K1. The sections of CHO/PA16-hipPDPN (A) and CHO–K1 (B) were treated with 0.1 μg/mL of PMab-322. The staining was carried out using BenchMark ULTRA PLUS with the ultraView Universal DAB Detection Kit. Scale bar = 100 μm.

Supplementary Fig. S1

The quantification of the PMab-322 reactivity in CHO–K1 cells, which overexpress PDPN derived from various species. The values (geometric mean of PMab-322 / geometric mean of each control mAb, Fig. 3) were determined.

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