Files
Abstract
Many biological events involve carbohydrates binding to protein receptors (lectins, antibodies, enzymes, etc). The carbohydrates are often present as glycoproteins or glycopids that may be free in solution or anchored to membranes. The strengthes of such interactions have been studied and reported using various techniques in the past, for instance: surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), biolayer interferometry (BLI), isothermal titration calorimetry (ITC), microscale thermophoresis (MST), nuclear magnetic resonance spectroscopy (NMR), and more qualitatively by glycan array screening. However, among all these techniques the ones involving surface immobilization such as SPR, ELISA and BLI have high sensitivity and are require low amounts of reagents, but often are unable to generate monomeric affinity measurement due to the typically multimeric nature of the glycans or the receptors. Those techniques that do not require any immobilization such as NMR, ITC and MST can measure the monomeric affinity values, but are less sensitive and may require considerably more reagent, especially ITC and NMR. To understand the complicated relationships between monomeric affinity and multimeric avidity, a more convenient method for measuring affinity is urgently needed. This work introduced a Biolayer Interferometry competition assay for the monomeric solution KD determination. Firstly, this approach was tested on two well-studied cases: Erythrina cristagalli lectin (ECL) and Human influenza A/Hong Kong/1/1968 (X-31) H3N2 hemagglutinin with their ligands; resulting in good agreement with literature. Secondly, a survey of receptor conformational properties was presented. Results highly suggested that conformational entropy played a key role in defining specificity. Thirdly, our robust and accurate assay was applied to a current hot topic: pandemic influenza hemagglutinin-glycan interactions. This was the first time reporting solution KD values for several important HAs. Fourthly, the inhibitory ability of several novel potential HA inhibitors where determined using the BLI competitive assay were presented. Finally, a study was completed using BLI to measure the direct binding of antibody binding: anti-blood group antibody specificity was demonstrated.