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

We read with great interest the recent article by Garelja et al. evaluating the reliability of commonly used enzyme-linked immunosorbent assay (ELISA) kits for measuring calcitonin gene-related peptide (CGRP) and appreciate their efforts to highlight the potential pitfalls in neuropeptide quantification.1 We fully agree with the authors that greater transparency from manufacturers is needed regarding the specificity, sensitivity, and performance characteristics of antibodies and kits so that researchers can make fully informed decisions about what is being measured. Until such transparency becomes standard practice, rigorous internal validation of each assay remains essential. As we have published three articles using saliva as a biological matrix and one of the ELISA kits mentioned by Garelja et al.,2-4 we would like to highlight several aspects of their conclusions that merit clarification. In our research, we have consistently referred to CGRP as CGRP-like immunoreactivity (CGRP-LI), explicitly acknowledging potential cross-reactivity and the challenges in isolating a single isoform. This distinction has been reported throughout our publications to ensure transparency regarding assay limitations while preserving the biological and clinical relevance of the findings. Our studies began in 2018, and over the years, ELISA kits have evolved considerably, with changes in their manufacturing processes and antibody composition. Even within the same commercial brand, kits may differ because manufacturers can change the antibody clone or production lot, leading to variability in affinity, cross-reactivity, and target recognition. In addition, most kits did not specify which CGRP isoform they detect. Therefore, direct comparisons between past studies and currently available kits are inherently problematic, and broad conclusions about the reliability of historical data based solely on contemporary assays are not warranted. Importantly, our publications provided detailed methodological information, including sample collection procedures, storage conditions, assay protocols, and quality control measures, ensuring reproducibility and critical appraisal by the scientific community. Regarding the study by Garelja et al., we note that they evaluated only two ELISA kits without a stated rationale for excluding other commercially available CGRP kits5 that have been used in other studies. Restricting the analysis to just two kits raises concerns about the external validity of their conclusions, given the well-documented variability among assays. Furthermore, while they rely on manufacturer validation of antibodies, they simultaneously criticize the use of these kits for actual CGRP measurement, which introduces an apparent inconsistency. Moreover, their assays also employed synthetic CGRP diluted in saline, which does not adequately replicate the biological milieu in which CGRP is normally present. In contrast, our measurements are conducted in saliva. Several studies have confirmed the presence of CGRP in this complex biological matrix,6, 7 supporting both its detectability and clinical relevance. Although no direct head-to-head study has conclusively demonstrated lower proteolytic degradation of CGRP in saliva compared with serum, several factors support this possibility. Saliva contains a rich protein milieu, including antiprotease factors and matrix components that can influence peptide stability and detectability.8, 9 In addition, studies have consistently reported higher CGRP concentrations in biological matrices such as saliva or tear fluid,10 sometimes exceeding those observed in plasma or serum. Collectively, these findings indicate that saliva may provide a distinct and often advantageous matrix for the sustained measurement of CGRP. Consequently, results derived from saline-based spike-in experiments may not fully reflect the behavior of endogenously released peptide within salivary secretions. Another important consideration is that the inability to quantify the final mature form of CGRP does not preclude detection of immature or intermediate forms, which may retain biological activity or serve as meaningful biomarkers. The relevance of these forms remains an important and unresolved question. Comparable methodological challenges are found in other research fields; for example, in Alzheimer's disease, beta-amyloid has been widely used as a biomarker despite known limitations related to isoforms and post-translational modifications.11, 12 Although rigorous internal validation of commercial kits is not uniformly performed across studies, collaborative efforts have enabled these biomarkers to make a meaningful impact on clinical research and currently have practical implications for diagnostic and therapeutic purposes. Our own work has demonstrated the clinical utility of salivary CGRP-LI through longitudinal monitoring of migraine attacks, correlation with clinical variables, phenotypic characterization, and evaluation of treatment response to anti-CGRP therapies, such as erenumab.2-4 These studies underscore the translational relevance of CGRP-LI measurement in saliva as a minimally invasive biomarker in precision medicine in migraine. Importantly, other groups have also employed the same commercial kit in different biological matrices, including serum,13 plasma,14 and even tear fluid,10 further supporting its broader applicability and reinforcing the concept that, despite methodological limitations, such assays can provide valuable biological and clinical insights when used with transparency and rigor. Moreover, our observations align with prior evidence showing elevated CGRP levels during migraine attacks and higher baseline concentrations in patients with chronic migraine, supporting both the biological plausibility and reproducibility of CGRP-related fluctuations in migraine pathophysiology.15, 16 In summary, while Garelja et al. rightly emphasize the technical challenges of neuropeptide quantification, their findings should not be interpreted as undermining the validity of carefully conducted studies using salivary CGRP-LI in migraine. We believe that critique alone is insufficient; constructive efforts to improve methodologies—similar to what has been achieved in other neurological diseases—are equally essential to advance the discipline. Methodological rigor, transparent reporting, awareness of matrix effects, and internal validation remain critical to advancing the field. Salivary CGRP-LI continues to provide valuable insights into migraine pathophysiology and treatment response. By performing comprehensive internal validation in future studies, we aim to further consolidate the utility of this biomarker, while continuing to advocate for greater transparency from kit manufacturers. Alicia Alpuente: writing – original draft; methodology; conceptualization; supervision. Victor J. Gallardo: conceptualization; formal analysis; methodology; validation; visualization; writing – review and editing. Laila Asskour, Edoardo Caronna, and Marta Torres-Ferrus: writing – review and editing. Patricia Pozo-Rosich: conceptualization; supervision; writing – review and editing. No funding or sponsorship was received for this study or publication of this article. Alicia Alpuente, Victor J Gallardo, Laila Asskour, Edoardo Caronna, Marta Torres-Ferrus, and Patricia Pozo-Rosich declare that there are no conflicts of interest regarding the publication of this paper. No external or internal medical writing or editorial assistance was used in the preparation of this manuscript.