A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.
A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.
Shallal
A A
AA
Department of Pharmacy, University of Strathclyde, Glasgow, UK.
al-Mahdawi
S A
SA
Kolodziej
M P
MP
Converse
C A
CA
D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000032:analysis*; D001327:Autoimmune Diseases / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005136:Eye Proteins / Q000276:immunology*; D015151:Immunoblotting; D010786:Photoreceptor Cells / Q000276:immunology*; D011817:Rabbits; D012164:Retinal Diseases / Q000276:immunology*; D012168:Retinal Pigments / Q000276:immunology*; D012243:Rhodopsin / Q000276:immunology*; D012374:Rod Cell Outer Segment / Q000276:immunology*; D017299:Rod Opsins; D012967:Sodium Dodecyl Sulfate
D001323:Autoantibodies; D005136:Eye Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; D012967:Sodium Dodecyl Sulfate; D012243:Rhodopsin