Genetic immunization with LYVE-1 cDNA yields function-blocking antibodies against native protein.
LYVE-1 is a surface bound hyaluronic acid (HA) receptor that is preferentially expressed by lymphatic endothelial cells (LEC). cDNA encoding full-length human LYVE-1 was coated onto gold particles that were then delivered via helium-assisted jet propulsion (gene gun) into the skin of Balb/C mice. LYVE-1 antisera, but not control pre-immune sera, recognized LYVE-1-transfected 293T cells by flow cytometry. While 40-70% of cultured human dermal microvascular endothelial cells (HMEC) were positive for LYVE-1 staining, human lung microvascular endothelial cells (LMEC) were negative. LYVE-1 antisera was used to effectively separate HMEC into LYVE-1 (hi) and LYVE-1(lo) populations that were enriched or depleted, respectively, for podoplanin, another LEC marker. By immunohistochemistry, LYVE-1 antisera detected CD31(lo) podoplanin(hi) lymphatic channels in normal and psoriatic human skin as well as in human tonsil. LYVE-1 antisera also blocked binding of FITC-labeled HA to HMEC (but not LMEC), demonstrating that these antibodies recognized regions of LYVE-1 required for HA binding. In summary, gene gun-assisted delivery of cDNA encoding LYVE-1 into skin resulted in reliable production of antisera that specifically and functionally recognized native LYVE-1 protein.
Cardones, AR; Leitner, WW; Fang, L; Murakami, T; Kapoor, V; Udey, MC; Hwang, ST
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