Enzyme-linked immunosorbent assay for quantitative determination of the visual pigment rhodopsin in total-eye extracts

Abstract

A versatile, multispecies enzyme-linked immunosorbent assay for the rod visual pigment (rhod)opsin has been developed. For this quantitative inhibition assay a monospecific polyclonal antiserum is used which is elicited in rabbits against bovine rod outer-segment membranes. Detergent concentrations as high as 1.0% can be used in the assay with only a slight loss in sensitivity. The assay allows quantitative determination of the apoprotein opsin with a detection level of about 0.04 pmol per sample by using standards prepared by illumination of spectrophotometrically determined amounts of rhodopsin. The antiserum shows considerable cross-reactivity with opsin from several species (mouse, rat, quail, monkey and man). The high degree of monospecificity and cross-reactivity of the antiserum already allowed quantitation of opsin content in crude eye extracts of mouse, rat and quail with a sensitivity comparable to that of bovine opsin. Similar types of multispecies immunoassays for quantitation of highly conserved membrane proteins can be developed using the described approach, requiring only a monospecific antiserum elicited against an easily accessible species and crude tissue extracts both for coating and as a source of the inhibitory antigen.