Version 1
: Received: 30 April 2024 / Approved: 1 May 2024 / Online: 1 May 2024 (09:02:09 CEST)
How to cite:
Gasson, R.; Roper, J. A.; Slack, R. J. A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors. Preprints2024, 2024050070. https://doi.org/10.20944/preprints202405.0070.v1
Gasson, R.; Roper, J. A.; Slack, R. J. A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors. Preprints 2024, 2024050070. https://doi.org/10.20944/preprints202405.0070.v1
Gasson, R.; Roper, J. A.; Slack, R. J. A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors. Preprints2024, 2024050070. https://doi.org/10.20944/preprints202405.0070.v1
APA Style
Gasson, R., Roper, J. A., & Slack, R. J. (2024). A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors. Preprints. https://doi.org/10.20944/preprints202405.0070.v1
Chicago/Turabian Style
Gasson, R., James A Roper and Robert J Slack. 2024 "A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors" Preprints. https://doi.org/10.20944/preprints202405.0070.v1
Abstract
Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was hemagglutination of red blood cells. Although insightful this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study we aimed to validate a more precise and quantitative method to enable further investigation of differences between galectins in respect to agglutination induction in different blood groups as well as characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer a more comparable effect was observed between blood group B and O but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration-responses and expected selectivity profiles for a set of small molecule glycomimetics, confirming historical profiles obtained in biochemical binding and functional cellular assays.
Keywords
Red Blood Cell Agglutination; Galectin-3; Galectin-1; Assay Optimisation
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
