Competitive ELISA for VGF Quantification

Competitive ELISA was performed through VGF assays previously used with human and mouse tissues [18 (link)]. Multiwell plates (Nunc, Milan, Italy) were coated with the relevant synthetic peptides and treated with PBS (containing 9% normal serum from the secondary antisera donor species, 20nM aprotinin, and 1mg/ml EDTA) for 2 hours. Primary incubations, with either human or rat/mouse VGF C-terminus antiserum, were carried out in duplicate, including serial standard dilutions in parallel with samples from human (plasma, fibroblasts) or mouse (spinal cord, plasma). Biotinylated secondary antibodies (Jackson, West Grove, PA, USA), streptavidin-peroxidase conjugate (Biospa, Milan, Italy), and tetrametylbenzidine (TMB X-traKem-En-Tec, Taastrup, Denmank) as substrate were used to reveal the positive labelling. Hence, the reaction was stopped with HCL (1mol/L) and the optical density was measured at 450nm using a multilabel plate reader (Chameleon: Hidex, Turku, Finland). Recovery of synthetic peptide/s added to plasma, or to tissue samples at extraction was >85% for all assays used. Each VGF assay was characterized using various synthetic peptides. Inter (CV1) and intra (CV2) assays were 3–4% and 9–11%, respectively. Data were expressed as mean ± SEM throughout.