Carboxymethyllysine levels and anti-carboxymethyllysine antibody levels measured by surface plasmon resonance in patients with age-related macular degeneration

Abstract

Purpose: Oxidative protein modifications such as N^ε-carboxymethyllysine (CML) have been associated with the development of advanced age-related macular degeneration (AMD). Serum biomarker levels can be used together with genotyping to calculate individual risk for development of advanced AMD, thereby enabling targeted implementation of preventative measures. Current methods for quantification of serum biomarkers involve either multiple time-consuming liquid chromatography steps or utilizing ELISA, which is an indirect measurement requiring sequential labeling/visualization steps. Surface plasmon resonance (SPR) has not previously been used to quantify serum biomarkers for AMD and has great potential to provide rapid, reproducible, accurate quantification.

Methods: Anti-CML antibodies (R and D systems, MN, USA) and CML-BSA (Cell Biolabs, Inc. CA, USA) in 10 mM sodium acetate, pH 4.5-5.0 were immobilized on the COOH1 sensor chip (SensiQ technologies Inc, OK, USA) using standard amine coupling. Serum samples were diluted in HEPES buffer pH 7.4 with 0.05% Tween and 1 mg/ml non-specific binding reducer (GE, USA). To dissociate native autoantibody-ligand complexes, serum was incubated in 300 mM acetic acid for 1 hour. Measurements were recorded on a SensiQ SPR instrument (SensiQ Technologies, Inc, Oklahoma City, OK) at 25⁰C.

Results: Levels of CML and anti-CML antibodies in unprocessed serum were successfully quantified using SPR with good reproducibility and minimal non-specific binding. As a proof of principle, serum from 7 patients with advanced AMD and 7 age-matched control patients were studied. The average level of CML in the patients with AMD was 45.6 +/- 11.3 response units (RU) (95% CI), whereas the average level of CML in the control patients was 34.0 +/- 10.5 RU. This difference was not statistically significant (p = 0.20). The average level of anti-CML antibodies in patients with AMD was 48.2 +/- 8.6 RU, which was similar to the level of anti-CML in control patients, 49.4 +/- 13.1 RU (p = 0.89).

Conclusions: SPR was utilized for the first time to successfully quantify an AMD-associated protein oxidation product and related autoantibodies. Establishment of this method permits future studies to characterize multiple serum biomarkers in large sample sizes.