title: A novel immunoassay for the evaluation of rod outer segment digestion in cultured retinal pigment epithelial cells.
abstract: In this work a novel enzyme-linked immunoabsorbent assay quantifying residual rod outer segments in the medium of rod outer segment-challenged retinal pigment epithelial cells is described. A retinal pigment epithelial cell line (D407) that produces low level of cathepsin D, and a primary human retinal pigment epithelial cell culture (HRPE51) that has normal cathepsin D levels, were challenged with bovine rod outer segments. At 3 days post-challenge, the amount of undigested or residual bovine rod outer segments left in the culture medium was quantified by an enzyme-linked immunoabsorbent assay. An antibody raised against bovine rod outer segments, which had been purified and labelled with nitroiodophenyl haptens, was used in the assay. The sensitivity of the immunoassay was less than 10(2) bovine rod outer segments per mL and the signal followed a linear curve, saturating around 10(6) bovine rod outer segments per mL. HRPE51 cells had no residual bovine rod outer segments present in the medium following a challenge with 10(4) bovine rod outer segments per mL. In the medium of D407 cells, residual bovine rod outer segment levels were higher at all bovine rod outer segment concentrations when compared to the residual bovine rod outer segment levels in HRPE51 cells, suggesting that D407 cells have a lower digestive capacity. These results demonstrated that the immunoassay for detecting bovine rod outer segments is a sensitive and reliable technique that can be used to quantify the amount of residual bovine rod outer segments, following bovine rod outer segment challenge of retinal pigment epithelial cells.
authors.affiliation: Centre for Ophthalmology and Visual Science, University of Western Australia, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: M C
authors.initials: MC
authors.lastname: Lai
authors.forename: I J
authors.initials: IJ
authors.lastname: Constable
authors.forename: P E
authors.initials: PE
authors.lastname: Rakoczy
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology; D010587:Phagocytosis / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000201:enzymology / Q000502:physiology*; D011817:Rabbits; D012374:Rod Cell Outer Segment / Q000276:immunology / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D002402:Cathepsin D