We have read the response letter with interest and agree with many of the points raised by the author(s).1 We welcome debate on the topic of neuropeptide measurement. For example, we agree that the use of calcitonin gene-related peptide (CGRP)-like immunoreactivity (CGRP-LI) is useful terminology. However, there are some points that require a response from us, to provide clarification on certain matters. First, our rationale for evaluating two particular enzyme-linked immunosorbent assay (ELISA) kits is explained in our original publication.2 To reiterate, our work originally stemmed from studies reporting that the Cusabio CSB-E08210h kit (Kit A) was selective for βCGRP over αCGRP.3, 4 We and others have a keen interest in understanding the differences between the two CGRP isoforms.4-8 Due to the high amino acid sequence similarity between mature αCGRP and βCGRP, generating antibodies that can truly distinguish between them is difficult. Kit A was therefore an interesting candidate for further research. As a first step, we needed to understand the selectivity profile of Kit A, using another kit that explicitly detects mature forms of both αCGRP and βCGRP (Bertin A05481; Kit B) as a control. It was never our intention to conduct an extensive experimental comparison of ELISA kits, although Table S1 in the Supporting Information of our original article may be of some value to readers.2 Nevertheless, our findings highlight the need for such work, and we wrote the publication such that others could consider using a similar approach as part of their validation efforts. Clarification of our use of mature synthetic CGRP to test these kits is important. This synthetic CGRP has the same sequence and biochemical activity as biologically-produced CGRP and has been used to understand basic CGRP biology for decades. We agree that the simple diluent used does not represent the complex biological milieu associated with patient samples such as saliva or plasma. However, our goal was simply to verify, under the simplest conditions possible, whether each kit could detect the mature bioactive form of each peptide, and the degree of cross-reactivity between the forms of CGRP. This is a necessary starting point toward understanding what each kit detects, given that biological matrices contain proteases and other factors that can otherwise confound measurement.9, 10 Once detection is established under carefully controlled conditions, a deeper investigation of the analyte in different biological matrices and how these may affect detection can then be conducted. For example, a recommended step in ELISA method development/validation is spike-and-recovery experiments, where a known quantity of analyte—for example, CGRP—is spiked into different matrices (or different dilutions of the matrix) to understand how much the matrix interferes with detection.9-11 Our study does not invalidate previously published results with Kit A, but it may alter their interpretation.11-16 This is because although manufacturer information and previous literature imply that this kit detects the mature βCGRP, in our hands we were unable to detect mature αCGRP or βCGRP, or define the actual analyte detected by this kit. As raised in our original publication, and as highlighted in the response letter, this kit may detect immature or intermediate forms of CGRP, generated during biosynthesis (although this is yet to be proven). Procalcitonin and mid-regional proadrenomedullin, both being intermediates of peptides belonging to the same peptide family as CGRP, are proposed as biomarkers for conditions including sepsis and/or bacterial infections.17, 18 Irrespective of the hypothetical value of measuring alternate forms of CGRP, it is essential to know which a particular kit detects, such that users can understand the results and ensure that like-for-like comparisons can be made between studies. It is likely that some of the literature discrepancies associated with CGRP measurement are attributable to different research groups detecting different forms of CGRP because they have used different ELISA kits. We do agree on the importance of transparency in research and tool development reporting. Suppliers should be as open as possible with data obtained during ELISA development, and researchers equally must be transparent when it comes to sharing their data using these kits. New guidelines, alongside more stringent journal requirements, aim to help ensure that all necessary methodological information is disclosed in reports.19 This includes listing the exact kits used in experiments (including catalogue numbers, batch numbers, and supplier details), the sample collection steps (including information about tubes used for collection and storage details), the steps taken during the ELISA protocol (such as sample preparation), and the data handling/manipulation that has occurred to arrive at the end result.9, 10, 16, 19 Additionally, it is important to report the rationale behind methodological decisions, to help readers understand why a certain approach has been taken. For example, if patient samples were diluted, it is important to explain why. Although many publications provide a good level of detail, many equally miss key elements, meaning that there is not enough information to enable full replication or interpretation by others. In some extreme cases, publications do not give enough information to even determine which kit(s) have been used, meaning that it is impossible to properly interpret the results. Although our original publication focused on the ability of ELISA kits to detect CGRP, it is important to stress that all tools based on antibodies require careful validation. There is now a wealth of evidence showing that antibodies are prone to having issues such as failing to detect the target protein, detecting off-target proteins, and having batch-to-batch variability, and so it is important to test antibodies to ensure they are valid to use.20-24 In this regard, we are unclear as to the meaning of the sentence, “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,” and so we cannot comment further on that statement. However, our position is that it is important to validate tools in your own laboratories under your own conditions before proceeding to use them in experiments. Well-validated tools are the cornerstone of reproducible research, and understanding their limitations is integral to properly interpreting experiments. Michael L. Garelja: Writing – original draft. Tayla A. Rees: Writing – review and editing; writing – original draft. Debbie L. Hay: Writing – review and editing. There are no conflicts of interest relevant to this letter for Debbie L. Hay, Michael L. Garelja, and Tayla A. Rees. None of the authors holds any interest in the manufacture or supply of CGRP ELISA kits. Debbie L. Hay is or has been a consultant or speaker for AbbVie, Nxera Pharma, Lilly, Amgen, Lundbeck, and Teva, and has received research funding from Pfizer, AbbVie, and Solros Therapeutics in the past 3 years. Debbie L. Hay is a member of the International Union of Pharmacology Transparency & Reproducibility Committee.
Garelja et al. (Thu,) studied this question.