Time Course for CEP Antibody Development and C3d Deposition in Bruch’s Membrane Following Immunization With CEP-MSA

Abstract

Purpose: : To study the development of CEP antibody and the presence of C3d over a time course of 7-90 days in our recently developed mouse model for age-related macular degeneration (AMD). C3d is a degradation product of C3b, a key protein required for the generation of the C3-C5 convertases in the classical, lectin and alternate pathways.

Methods: : During the 7-90 day time course, mice were immunized with CEP/MSA in complete Freund’s adjuvant (CFA) or CFA alone at day 0, followed by a challenge at day 10 in incomplete Freund’s adjuvant, except the day 7 mice which received only the initial immunization of 200µg CEP-MSA. To generate a strong immunological response, a second boost was given 10 days before the mice were sacrificed. Following enucleation, eyes were place in OCT embedding media. For blood analysis, blood was obtained from the ocular sinuses and centrifuged for the collection of plasma. ELISA testing was performed against plates coated with MSA for the quantification of CEP antibody levels. Cryosections 7µ n thickness were prepared on a Cryostat HM 505E (Microm). For C3d localization, a polyclonal rabbit anti-human antibody C3d antibody was applied at a 1:100 dilution, followed by the secondary antibody (swine anti-rabbit immunoglobulin/FITC). Slides were mounted with Vectashield and viewed for fluorescence.

Results: : CEP antibody levels in the short-term recovery animals were 6-8 times higher in the CEP-MSA immunized mice compared to naïve and control mice immunized with CFA. C3d was observed in Bruch’s membrane below the RPE in the mice receiving CEP-MSA immunization. Although the C3d immunofluorescence was somewhat patchy in eyes from CEP-immunized mice, it was not observed in control mice.

Conclusions: : Ongoing studies indicate that antibody titers and C3d immunofluorescence increase over the length of time. R.Venkatesh and T. Yamamoto are co-first authors.

Keywords: Bruch's membrane • immunohistochemistry • age-related macular degeneration