| Autor: |
Krishnan PS; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine; Virginia Commonwealth University School of Medicine., Zamuner FT; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine; Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine., Jenks CM; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine., Xie JY; Department of Otolaryngology-Head and Neck Surgery, University of Michigan Medical School., Zhang L; Department of Otolaryngology-Head and Neck Surgery, The Ohio State University College of Medicine., Lehar M; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine., Fedarko NS; ICTR Clinical Research Core Laboratory, Johns Hopkins University School of Medicine., Brait M; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine; Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine., Carey JP; Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine; jcarey@jhmi.edu. |
| Abstrakt: |
Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role. |
