Galanin was shown to play an important role

Galanin was shown to play an important role in pain and pain processing [14] and the involvement of neuropeptides in the development of migraine is well established [[24], [25], [26]], however, so far it has not been examined whether systemic galanin levels correlate with migraine. Therefore, we used the in-house ELISA to detect galanin in the plasma of migraine patients and healthy controls.
Materials and methods
Results
Discussion Antibodies have become an indispensable research tool and have also been developed as therapeutic agents. However, scientific progress is hindered by Domiphen Bromide that are marketed as being specific but in fact lack specificity. Although this issue has been persistently addressed in the literature [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12],38,39], data on the validation process of antibodies and antibody-based tools are seldom seen in reports applying these tools [[40], [41], [42]]. This is of critical importance, because researchers new to a field tend to use antibodies that have been employed in previously published studies. Moreover, validation of commercial antibodies by the suppliers is often inadequate and unreliable, making validation by the user essential. An important aim of this article is to raise awareness of the necessity to validate antibodies and antibody-based tools when working in the galanin research field. There is a vast number of different commercial antibodies marketed at galanin researchers, with antibodypedia.com currently (April 2018) listing 244 antibodies against galanin, 257 against GAL1-R, 206 against GAL2-R, and 178 against GAL3-R (independent of reactivity) being sold by up to 33 different vendors. Some of these are listed in Supplementary Tables S1–S4, but we make no claim of completeness. We tested 18 commercial antibodies against galanin and its three receptors and identified specific antibodies against all four of those proteins, which we currently use for immunohistochemical analysis on human tissue samples [27,43] (specific antibodies are underlined in Supplementary Tables S1–S4). Unfortunately, we could not identify commercial antibodies specific for any of the three murine galanin receptors. Thus, the results of a recent study investigating changes in expression of galanin receptors in the rat heart upon acute immobilization stressors have to be treated with caution, since the authors did not evaluate the antibodies themselves, but stated that “the standard quality of all antibodies was checked [by the supplier]” [41]. Fourteen anti-human antibodies tested in this study were classified as unspecific with the techniques we applied; however, this does not necessarily mean they will lack specificity in other assays or under different test conditions. For example, antibody HPA044513 against human GAL2-R (Sigma-Aldrich) is being used by The Human Protein Atlas (www.proteinatlas.org), whereas it showed unspecific background staining in our immunohistochemical assays. Although there are numerous guidelines for proper antibody validation in the literature [[44], [45], [46], [47], [48], [49], [50]], we want to highlight four critical points: (1) Antibody specificity should be evaluated by several techniques, because a single technique may not be conclusive to assess the overall usefulness of an antibody. This is mainly attributable to the specific nature of antibodies; polyclonal antibodies generally are able to recognize different structural and/or sequence epitopes on the same molecule, whereas monoclonal antibodies are restricted to a single epitope. Clearly, this determines the possible application spectrum of a given antibody for biochemical analyses. For example, one antibody might not be suitable for western blot analysis but it may be a potent tool in immunohistochemistry, or vice versa. (2) Antibody results very much depend on the production lot, with great lot-to-lot variation being observed frequently for polyclonal antibodies. Therefore, each new lot of an antibody should be re-evaluated accurately before use. For example, we experienced that a new lot of a specific antibody against human GAL2-R did not show the same specificity as the previous lot and, consequently, the antibody was withdrawn from the market (LS-4251, LifeSpan). (3) Selection of appropriate positive and negative controls for the validation tests is crucial. Transfected cell lines are highly recommended. However, as reactivity depends on target concentration, it is not guaranteed that specificity toward a protein expressed at high levels will be maintained when the protein is expressed at low levels. The best negative controls are obviously KO animals or tissues with silenced expression, but access to such tissues may be difficult or, in the case of human tissue, impossible. Moreover, it is difficult to find human tissues lacking galanin expression. Therefore, correlation of protein expression data to mRNA expression levels is one way to solve the problem of proper controls. Finally, (4) validation data provided by the antibody suppliers should be evaluated skeptically and critically, especially if crucial information about the antigen is not accessible or even withheld. Proprietary reasons are often mentioned, but scientific work is hampered tremendously by these claims. Antibody specificity should be doubted if suppliers show multiple bands in immunoblot analysis (e.g. AP01217PU-N, Acris; ab59027, Abcam; EB07562, Everst Biotech; see Supplementary Tables S1-S4). Even antibody data sheets should be checked for accuracy regarding the cited literature. For example, Abnova references a publication from our lab for the use of their GAL2-R antibody PAB7174; however, that antibody was never used in the referenced study [51].