Analysis Biomolecular Interactions of HB36.5/HB36.6 with Hemagglutinin (HA) by BLI (CAT#: STEM-MB-0193-CJ)

Introduction

The influenza envelope glycoprotein hemagglutinin (HA) on the surface of the influenza virus consists of a highly variable globular head domain (HA1) and a more conserved stem domain (HA2/HA1). Influenza viruses comprise two phylogenetic groups (Groups 1 and 2) consisting of 18 HA subtypes and numerous genetic variants or strains within each subtype. Although vaccination can prevent influenza infection, current vaccines are strain specific, and provide minimal protection against drifted or shifted strains or subtypes.

Broadly neutralizing monoclonal antibodies (bnAbs) that bind the conserved HA stem can neutralize diverse influenza strains in vitro, suggesting that antivirals targeting the HA stem could provide similar widespread protection. BnAbs can bind to the fusogenic region of the HA stem and inhibit the conformational rearrangements in HA required for membrane fusion. The protection by HA-stem binding bnAbs is greatly enhanced through FcγR engagement in vivo. And Fc-FcγR interaction and activation of antibody-dependent cellular cytotoxicity (ADCC) are critical for in vivo efficacy of stem-binding bnAbs.

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Principle Applications Procedure Materials Notes Related Products

Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Immunology/Inflammation; Virology; Pharmacology

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: QiaQuick Kit

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