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Peter Baciu, Maria Burton, Jae Yang, Justine J Cunningham; Qualification of a Multiplex Panel to Analyze Complement Proteins in Human Ocular Tissues. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2301.
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© ARVO (1962-2015); The Authors (2016-present)
Genetic dysregulation of the complement cascade has been linked to an increased risk for the development of age related macular degeneration (AMD). The complexity of the complement cascade together with the lack of multiplex tools, makes it difficult to elucidate the key interacting proteins that underlie the pathogenesis of AMD. Our goal was to qualify a multiplex complement panel as a tool for future determination of complement proteins associated with ocular disease in donor eyes.
Six pairs of human eyes (NDRI, NY) from a random population sample, were dissected and tissues processed following our previously optimized protocol. Briefly, vitreous samples were diluted 1:1 with PBS + protease inhibitors, passed through 23/25G needles, centrifuged (12,500G/15 mins/4°C), and the supernatant passed over a 0.45µm PVDF Durapore column. Retina/choroid were separated, homogenized in TER-I lysis buffer (Life Technologies) + protease inhibitors. Homogenates were rested (1hr/4°C), centrifuged (12,500G/15 mins/4°C), and supernatant collected. Pooled normal human serum from Quidel (San Diego, CA), and complement proteins from CompTech (Tyler, TX) and Quidel, were used to benchmark the assay and to assess assay specificity, accuracy, linearity and matrix effects. Multiplex complement panels were purchased from EMD Millipore (Billerica, MA).
No matrix interference and good standard recovery (≥80%) was observed in the buffers used for tissue homogenization. Good spike recovery (CV ±20%) and assay linearity was observed for purified proteins (C5, C3, FD, FB, FP, FI, FH, MBL). Serum levels of the complement proteins approximated values from the literature and showed corresponding changes upon serum activation. Within the ocular tissues FB, FD and C3 showed similar levels to those reported in the literature. Ocular levels of C5, FP, FH and MBL were consistent with local synthesis. Six proteins – C1q, C2, C4, C4a, C5a, and C9 – did not meet our assay criteria for specificity, accuracy, linearity and matrix effects.
We qualified a multiplex complement panel for use in ocular tissues and provide the first report for levels of C5, C3, FD, FB, FP, FI, FH, MBL in donor human eyes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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