Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Abstract

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

Epitope Mapping of Anti-Tiger Podoplanin Monoclonal Antibody PMab-231

“…Conversely, the binding epitopes of mAbs produced by immunizing with synthesized peptides have been easily determined using deletion mutants and point mutants of synthesized peptides. (29,30) We first synthesized a series of tigPDPN peptides, which are summarized in Table 1. Using ELISA, PMab-231 detected 24-43 peptides corresponding to the 24th-43rd amino acids (aas) of tigPDPN.…”

Epitope Analysis of an Anti-Whale Podoplanin Monoclonal Antibody, PMab-237, Using Flow Cytometry

“…(22,34,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) These epitope mapping results showed that almost all anti-PDPN mAbs react with PLAG domains or PLDs. (7,(39)(40)(41)43,44,(49)(50)(51) The critical epitope of PMab-237 was also shown to be located in PLD (Fig. 4), suggesting that PLAG domains and PLD were advantageous to the epitope for several applications such as flow cytometry, western blotting, and immunohistochemical analyses.…”

Epitope Mapping of the Anti-California Sea Lion Podoplanin Monoclonal Antibody PMab-269 Using Alanine-Scanning Mutagenesis and ELISA

“…(31,32) We have established different anti-PDPN mAbs for many species by employing a cell-based immunization and screening (CBIS) method, and characterized the targeted epitopes. (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47) Recently, we developed a novel anti-PDPN mAb, clone PMab-269 (mouse IgG 1 , kappa), which recognizes the California sea lion (Zalophus californianus) PDPN (seaPDPN). (48) The PMab-269 clearly detected seaPDPN protein in flow cytometry and Western blotting analysis.…”