Calcitonin gene-related peptide and headache: Comparison of two commonly used assay kits highlights the perils of measuring neuropeptides with enzyme-linked immunosorbent assays.

OBJECTIVES/

backgroundThis study was undertaken to test the ability of a widely used enzyme-linked immunosorbent assay (ELISA) kit to detect calcitonin gene-related peptide (CGRP) isoforms to better understand currently published clinical data. There is significant interest in measuring CGRP as a biomarker in headache and migraine research, with ELISAs being the preferred detection method. ELISAs use antibodies that have been raised against an antigen to allow selective quantification of an analyte in a sample. Understanding the specificity of these antibodies is crucial to interpreting results. One commercially available kit (Cusabio CSB-E08210h, Kit A) is purported to specifically detect β-CGRP (one of the two CGRP isoforms) over α-CGRP and has been used to investigate the potential for CGRP to be used as a biomarker for migraine. We investigated the ability of this kit to detect multiple isoforms of CGRP to better interpret published clinical results. We used a second ELISA kit (Bertin Bioreagent, A05481, Kit B) that is nonselective for the different CGRP isoforms as a control to ensure the CGRP we used could be detected in ELISAs.

methodsWe performed ELISAs according to the manufacturer's instructions, testing concentrations within the advertised range of each kit. At least three independent experiments were conducted for each kit.

resultsKit B was able to detect both human and mouse α-CGRP and β-CGRP with a high degree of reproducibility. In contrast, Kit A did not detect bioactive forms of human α-CGRP or β-CGRP, nor mouse α-CGRP or β-CGRP.

conclusionKit A may not be reliable for future studies, as it does not appear to detect mature bioactive CGRP. Importantly, conclusions from previous studies that used this kit may need to be reevaluated, as it is not clear what analyte the kit has detected. Our findings also highlight the importance of understanding research tools to ensure accurate interpretation of results.