Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • Introduction Since the development of the first radioimmunoa

    2022-05-16

    Introduction Since the development of the first radioimmunoassay (RIA) to measure hormone levels in the late 1950s, for which Rosalyn Sussman Yalow was awarded the Nobel Prize for Medicine in 1977 (the second woman ever receiving it), the use of PHA-665752 sale in molecular research has increased tremendously. Over the decades, antibodies have become a versatile tool and are used, for example, to qualitatively and quantitatively measure polypeptides, to elucidate the cellular and subcellular distribution of proteins, to sort cells, or to reveal post-translational modifications such as phosphorylation, acetylation, or ubiquitination. Furthermore, antibodies have been used as therapeutics since the mid-1980s, when the first monoclonal antibody was approved by the US Food and Drug Administration to prevent the rejection of renal transplants [1]. The expansive use of antibodies, however, is accompanied by substantial controversies. An inadequate validation process or – even worse – no validation at all, may give rise to false-positive or false-negative results leading to the inability to replicate previous observations [[2], [3], [4], [5]]. Clearly, research is hampered by the lack of specific antibodies. The specificity of antibodies is especially crucial when they are applied to study proteins with low expression levels, as it is the case in endocrinology, an issue already widely addressed [[6], [7], [8], [9], [10], [11], [12]]. An illustrative example of the necessity of antibody specificity concerns the regulatory (neuro)peptide galanin. Galanin and its three endogenous G-protein-coupled receptors (GAL1-3-R) are widely distributed throughout the central and peripheral nervous systems as well as in non-neuronal peripheral tissues. However, in general, cells/tissues with high expression levels are rare, especially under healthy baseline conditions [13,14]. Typical for neuropeptides, galanin is derived from a larger peptide precursor (pre-pro-galanin, 123/124 aa) and proteolytic cleavage yields the mature 29 aa/30 aa (in humans) peptide. Galanin is processed along with the 59 aa galanin message-associated peptide (GMAP) from the same precursor. This complicates the development of anti-galanin antibodies, because they should be specific only for the functionally mature galanin peptide but should not detect GMAP. Thus, antibody targets which are designated as anti-“pre-pro-galanin” usually do not specify what they detect – precursor only, GMAP and galanin, only GMAP or only galanin? Adding to the complexity of antibody development and epitope selection for galanin is the homology of galanin’s first 13 aa to aa 9–21 of the galanin-like peptide (GALP) (Table 1), which originates from another gene. The small peptide alarin, which is a splice variant of GALP [15], does not share sequence homology with galanin. The situation is similarly complex for the three galanin receptors, as they share 38% (GAL1-R vs. GAL3-R), 42% (GAL1-R vs. GAL2-R), and 53% (GAL2-R vs. GAL3-R), respectively, aa sequence homology in humans [14,16]. Thus, careful validation of commercially available antibodies against galanin receptors is essential, which is underscored by the findings of two independent groups, who reported similar immunohistochemical staining between tissues of wild-type and galanin receptor knockout (KO) mice using various commercial antibodies [17,18] – clearly an unacceptable result. Therefore, we tested the specificity of several commercially available antibodies against human and mouse galanin as well as all three galanin receptors and report here which of them we could validate and recommend for use. No less caution should be used in case of antibody-based tools, like RIA and enzyme-linked immunosorbent assay (ELISA). The validity of commercial galanin RIA kits was previously established [19] and they are now being used routinely [[20], [21], [22], [23]]. However, we observed that the same does not hold true for commercially available human galanin ELISA kits. Thus, we tested four different sandwich ELISA kits from different vendors, but the validation data were disappointing. Therefore, we developed and validated a sandwich ELISA for human full-length galanin.