Abstract
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.
Highlights
In many studies, alpaca has been used for production of antigen-specific single domain antibodies [1,2,3]
Stable transfectants were selected by limiting dilution and cultivating in a Abbreviations: CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary; CLEC-2, C-type lectin-like receptor-2; DAB, 3,3′-diaminobenzidine tetrahydrochloride; alpaca PDPN (aPDPN), alpaca podoplanin; human PDPN (hPDPN), human podoplanin; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PDPN, podoplanin; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate
Two mice were immunized with CHO/aPDPN cells (Fig. 1)
Summary
Alpaca (lama pacos) has been used for production of antigen-specific single domain antibodies (nanobodies) [1,2,3]. The type I transmembrane glycoprotein, podoplanin (PDPN)/T1alpha/Aggrus, is expressed in normal tissues, including type I lung alveolar cells, renal podocytes, and lymphatic endothelial cells [4,5,6]. The expression of human PDPN (hPDPN) has been reported in several malignant tumors, including malignant brain tumors [14,15,16,17], malignant mesotheliomas [18,19], oral squamous cell carcinomas [20], esophageal cancers [21], lung cancers [22], osteosarcomas [23,24,25], chondrosarcomas [24], and testicular tumors [26]. The expression of hPDPN is associated with malignant progression and cancer metastasis [9,14,27]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
