Quantibody human angiogenesis array 1000

Sixty angiogenic and inflammatory peptides were quantified with a multiplexed sandwich ELISA-based quantitative protein microarray (Quantibody Human Angiogenesis Array 1000, RayBiotech, Norcross, Georgia). Capture antibodies for 60 human peptides were adhered to wells on a glass plate. Plasma samples were diluted 1:1 in a physiologic buffer and incubated with capture antibodies for 2 h at room temperature. Sample wells were washed, and 60 biotinylated detection antibodies were added. After washing, a streptavidin-labeled Cy3 fluorescent probe was added. Peptide quantification was performed by the manufacturer with a laser scanner (GenePix 4100A, Molecular Devices, San Jose, California), GenePix Pro7 (Molecular Devices), and Q-Analyzer Software (Raybiotech). Peptide levels were interpolated from standard curves.

The Quantibody Human Angiogenesis Array 1000 (RayBiotech Inc., Norcross, GA, USA) was used for analysis of 60 angiogenesis‐related proteins (supplementary material, Table S1). This array is based on the sandwich enzyme‐linked immunosorbent assay (ELISA)‐technology, and each antibody is spotted in quadruplicate. The procedure was performed at room temperature. First, all wells were blocked with sample diluent. Then, the array was incubated with serum samples and the standard dilutions for 2 h. The washing process was followed by incubation with the detection antibody for 2 h. After further washing, Cy3 equivalent was added to each well and incubated for 1 h. Imaging was performed by a microarray scanner (GenePix 4000B, Axon Instruments, San Jose, CA, USA) at different photomultiplier tube gains. Interslide normalisation for the most suitable scan was performed by RayBiotech (Norcross, GA, USA). The concentrations based on linear standard curves were used for further analyses.