Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2019
Publication Date: 2/21/2019
Citation: Zhao, W., Baldwin, E.A., Cameron, R.G. 2019. A digital data interpretation method for hemagglutination inhibition assay by using a plate reader. Analytical Biochemistry. https://doi.org/10.1016/j.ab.2019.02.016.
Interpretive Summary: The basis of hemagglutination and hemagglutination inhibition assay is the binding of carbohydrate-binding proteins found on the surface of influenza and several other viruses to chemical decorations on the surface of red blood cells. This binding results in a network, or lattice structure of agglutinated red blood cells, which maintain the red blood cells in a suspended distribution, typically viewed as a diffuse reddish solution. These assays are used not only for characterizing a wide range of viruses and the associated immune response, but also have been exploited to probe glycan-lectin interactions. The galectin family of proteins are beta-galactoside-binding lectins, they have diverse effects on many cellular functions in humans including adhesion, migration, polarity, chemotaxis, proliferation, apoptosis, and differentiation. Therefore, they play roles in many pathological states, including tumor cell metastasis, autoimmune diseases, allergic reactions, inflammation, atherosclerosis, and diabetic complications. Galectins also possess hemagglutination activity, by binding to and cross-linking structures located on the surface of red blood cells. Therefore, the hemagglutination inhibition assay provides a simple and quick method for studying binding activities of galectins with glycans or glycoproteins. The hemagglutination inhibition assay is challenging due to the inherent subjectivity between different readers of various experience levels. To overcome these limitations we have developed a digital data interpretation method for hemagglutination and hemagglutination inhibition assays by using a microplate reader which provides a objective numerical response.
Technical Abstract: The hemagglutination inhibition assay is a simple method for quantifying relative binding activities of glycan-lectin interactions. Currently, interpretation of hemagglutination inhibition assay data remains a manual task depending on the subjective visual interpretation of red color development in the assay solutions. In this study we developed a digital data reading method for the hemagglutination inhibition assay assay by using the area scanning function of a microplate reader. This was based on inhibition assays for galectin-3 induced hemagglutination. Optical density values showed a four-parameter logistic correlation with concentrations of galectin-3 inhibitors, and 50 percent inhibition concentration values were obtained from the curve fitting. The method provides an objective and robust data interpretation for hemagglutination inhibition assays conducted with chicken erythrocytes.