Purpose: Age-related macular degeneration (AMD) is the leading cause of severe vision loss among the elderly population worldwide. AMD has been shown to result from a chronic, aberrant inflammatory response, specifically involving the complement cascade. Numerous studies have suggested that localized disruption of the complement cascade results in arresting progression of AMD. PEGylated drug is an anti-PDGF-B PEGylated, stabilized aptamer targeted as a potent and selective inhibitor of factor C5 in the complement cascade. By inhibiting C5-mediated inflammatory activities, therapeutic benefit can be achieved in both dry and wet AMD. Numerous Phase I studies have been initiated to evaluate PEGylated drug both individually and as part of combination therapies. To support such studies, Syneos Health has developed a hybridized ELISA method for the detection of PEGylated drug in human plasma.
An amino microplate is coated with Capture Oligonucleotide. After thoroughly washing the plate to remove unbound capture nucleotide, the plate is blocked with blocking buffer. During the blocking step, the standards, QCs, and samples undergo heat inactivation. The PEGylated drug in K2EDTA human plasma is then hybridized with biotinylated Detection Oligonucleotide using a thermal cycler. After washing the blocked capture plate, the mixture of Biotinylated Oligonucleotide and PEGylated drug in standards, controls, and samples are added to the capture plate. The PEGylated drug in the standards, controls, and samples will bind to the Capture Oligonucleotide coated wells. Streptavidin Poly-HRP is used to detect the nucleotide/ PEGylated drug complex. Excess unbound conjugate is removed by further plate washing. The bound conjugate is visualized by adding the substrate tetramethylbenzidine (TMB) to the plate. Sulfuric acid is used to stop the TMB reaction.
A robust oligonucleotide hybridized ELISA has been developed to detect PEGylated drug in human plasma samples.