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    • In the present study we

    2021-10-14

    In the present study, we also found that specificity concerns persist for antibody-based tools, like ELISAs. Measuring the same human plasma sample with four different commercial galanin ELISA kits produced four widely different galanin concentrations. No recovery of synthetic galanin peptide added to human plasma was observed with all four ELISA kits, but even spiking human plasma with the supplied standard peptide achieved only poor recovery rates. In contrast, synthetic galanin was detected by all ELISAs if it was dissolved in supplied sample diluent, although, the amount of tested peptide was not determined correctly. Furthermore, all tested peptides were detected regardless of their homology to the galanin peptide. Most intriguingly, the galanin standard peptides supplied in the ELISA kits were not cross-reactive, as the peptide of any one ELISA kit was not adequately recognized by the other three kits. Although one of the four commercial galanin ELISA kits tested is no longer available (Abbexa, abx051368), we still implement it in our test results to highlight the need for critical testing of Ryuvidine and also antibody-based tools. Based on our investigations presented here, these commercially available galanin ELISAs could not be validated and the data generated do not support the use of these assays to measure galanin in plasma. Obviously, these findings cast doubt on published data using these ELISA kits [42,52,53]. Another way to confirm results obtained by ELISA or RIA might be a quantification of the peptide of interest in serum or tissue using mass spectrometry. This is the most accurate way to determine the concentration of such peptides with the additional advantage to also detect truncated versions and the ratio of the different fragments [54]. However, this method might not be available to all researchers. Unable to validate a commercial galanin ELISA kit, we developed a sandwich ELISA against human galanin using commercially available antibodies. The combination of a murine monoclonal antibody with a polyclonal rabbit antibody proved to be highly specific. This in-house galanin ELISA detects only the mature full-length galanin peptide but not related peptides, shows a satisfactory recovery rate for plasma samples spiked with synthetic galanin peptide, and is able to detect endogenously expressed galanin in supernatants of induced, galanin-overexpressing HaCaT cells. The measured galanin plasma levels match with published levels detected by RIA [[34], [35], [36], [37]]. Furthermore, this in-house ELISA is easy to perform and relatively inexpensive (<100 € per plate, as of April 2018). Most importantly, only 100 μl of plasma sample is needed and it can be applied directly without protein extraction. To our knowledge, the only other antibody-based assays capable of detecting endogenous and synthetic human full-length galanin in plasma samples are the RIA kits from Peninsula (S-2126.0001) and Phoenix (RK-026-01). However, those assays lack appropriate sensitivity and, therefore, proteins need to be extracted from plasma samples and concentrated. Thus, up to 10 ml of plasma is needed per patient. Furthermore, Grenbäck and colleagues showed that galanin plasma levels measured by RIA are heavily influenced by the extraction method applied [55]. After developing a specific galanin ELISA, we analyzed galanin plasma levels in migraine patients. The role of neuropeptides in the development of migraine is well established. Neuropeptides such as calcitonin-gene related peptide (CGRP) and substance P (SP) contribute to a migraine attack by triggering neurogenic inflammation in the nervus trigeminus in the brain, with subsequent edema formation and mast cell activation [24]. Similarly, these neuropeptides seem to be involved in cortical spreading depression (CSD), which contributed to the migraine aura [25,26]. Sensory spinal cord neurons expressing galanin were found to co-express CGRP and SP, and the role of galanin in pain, pain processing and nociception is well established [14]. Regarding migraine, it was shown that experimental CSD in rats resulted in long-lasting induction of galanin mRNA and peptide levels across the ipsilateral cerebral cortex in activated oligodendrocyte progenitor cells. At 7–28 days post CSD induction, an increase in GAL1-R mRNA levels was observed in the neocortex and the piriform cortex, accompanied by a loss of galanin-positive nerve fibers [56]. However, a direct link between galanin and migraine in humans has not been examined so far. In the present study, galanin plasma levels in migraine patients and healthy controls matches with published plasma levels detected by RIA [[34], [35], [36], [37]], however, we found that galanin plasma levels were not altered during a migraine attack, and migraine patients also exhibit similar galanin plasma levels in a pain-free interval compared to healthy controls. Furthermore, in the group of migraine patients, galanin plasma levels did not correlate with pain severity, migraine type (with or without aura) or age.