A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors

TL;DR

This paper introduces a more precise method to measure red blood cell agglutination caused by galectins, aiding in the study of galectin inhibitors for diseases like cancer.

Contribution

The study validates a quantitative hemagglutination assay using imaging and analysis with FIJI ImageJ for galectin characterization.

Findings

U-bottom plates with FIJI ImageJ imaging provided better quantification of red blood cell agglutination than optical density plate readers.

Galectin-3-induced agglutination was most effective in blood group B, while galectin-1 showed similar effects in B and O but stronger than in A.

Inhibition assays demonstrated concentration-dependent responses and selectivity profiles for small-molecule inhibitors.

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 the hemagglutination of red blood cells. Although it is 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 the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the 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 the historical profiles obtained in biochemical binding and functional cellular assays.

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