PcMab-47: Novel Antihuman Podocalyxin Monoclonal Antibody for Immunohistochemistry

Podocalyxin (PODXL) is a CD34-related sialomucin and a well-known marker of embryonic stem cells. PODXL is expressed in many types of tumors including colorectal cancers, breast cancers, and brain tumors. Overexpression of PODXL is an independent predictor of progression, metastasis, and poor outcome. PODXL is also expressed in many normal cells such as renal podocytes and endothelial cells (ECs). However, high-sensitive and high-specific anti-PODXL monoclonal antibodies (mAbs) have not been established. Herein, we immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells. The anti-PODXL mAb, PcMab-47, reacted with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in flow cytometry. Immunohistochemical analysis showed that PcMab-47 detected PODXL-expressing normal cells such as podocytes of kidney or ECs. Furthermore, PcMab-47 stained PODXL-expressing cancer cells of colon or breast cancers. These results suggest that PcMab-47 could be useful for investigating the expression and function of PODXL in cancers and normal tissues.


Hybridoma production
Four-week-old female BALB/c mice (CLEA, Tokyo, Japan) were immunized by intraperitoneal (i.p.) injection of the purified ectodomain of human PODXL (100 mg) together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster i.p. injection of LN229/PODXL was given 2 days before the mice were euthanized by cervical dislocation, and spleen cells were harvested. The spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). Hybridomas were grown in RPMI 1640 medium including l-glutamine with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to the purified ectodomain of PODXL. Proteins were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific, Inc.) at 1 mg/mL for 30 minutes. After blocking with 1% bovine serum albumin (BSA) in 0.05% Tween20/phosphate buffered saline (PBS; Nacalai Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:2000 diluted peroxidase-conjugated antimouse IgG (Agilent Technologies, Inc., Santa Clara, CA).

FIG. 2.
Immunohistochemical analysis of human kidney by PcMab-47. A section of kidney was incubated with 1 mg/mL of PcMab-47, followed by the Envision+ kit (A, B). Color was developed using 3, 3-diaminobenzidine tetrahydrochloride, and counterstained with hematoxylin. Another section of kidney was also stained using hematoxylin and eosin (C, D). Scale bar: 100 mm.
The enzymatic reaction was produced with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories Inc., Hercules, CA).

Flow cytometry
Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washing with 0.1% BSA in PBS, cells were treated with primary mAbs for 30 minutes at 4°C, followed by treatment with Oregon Green 488 goat antimouse IgG (Thermo Fisher Scientific, Inc.). Fluorescence data were collected using the Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Immunohistochemical analyses
Human normal tissues and cancer tissues were purchased from BioChain Institute, Inc. histological sections were deparaffinized in xylene and rehydrated. After the antigen retrieval procedure (autoclave using citrate buffer, pH 6.0; Agilent Technologies, Inc.), sections were incubated with 1 or 10 mg/mL of PcMab-47 for 1 or 3 hours at room temperature followed by treatment with Envision+ kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 5 minutes, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.).
Herein, we immunized mice with recombinant PODXL, which was purified from the culture supernatant of LN229/ ectodomain-PODXL. A booster i.p. injection of LN229/ PODXL was administered. The culture supernatants were screened using ELISA for binding to purified PODXL. As a second screening, we performed flow cytometry for reaction with LN229 and LN229/PODXL. A stronger reaction against LN229/PODXL than against LN229 was necessary, and one clone, PcMab-47 (mouse IgG 1 , kappa), was produced after limiting dilution.
Next, we investigated the immunohistochemical utility of PcMab-47 in normal tissues or cancers. As shown in Figure 2, PcMab-47 stained podocytes or ECs of kidney ( Fig. 2A, B). PcMab-47 reacted with colon adenocarcinoma, in which membrane/cytoplasmic-staining pattern was observed (Fig. 3A,  B). Strong expression of PODXL was also observed in ECs in colon adenocarcinoma (Fig. 3B). In contrast, weak expression of PODXL was observed in cancer cells, but stronger expression was detected in ECs in breast cancer (Fig. 3C, D). These results indicate that PcMab-47 is very useful for immunohistochemical detection of both cancer cells and normal cells that express PODXL.
Therefore, PcMab-47 may facilitate investigation of the expression and function of PODXL in cancer and normal tissues. Future investigations should include production of different epitope-possessing anti-PODXL mAbs to elucidate the function of PODXL.