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Analysis Kinetics of E3_53 to free IgE or IgE immobilized on FcεRIα by BLI (CAT#: STEM-MB-0286-CJ)
Introduction
Mechanistically, an overwhelming amount of clinical and experimental evidence indicates that the predominant form of human anaphylaxis is immunoglobulin E (IgE) dependent. The most common allergens inducing anaphylactic reactions are found in foods (e.g. peanuts, tree nuts, fish or shellfish), drugs (e.g antibiotics, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents) or animal venoms (e.g. insect stings, snake bites). Antigen-mediated cross-linking of allergen-specific IgE bound to the high-affinity IgE receptor (FcεRI) on mast cells and basophils induces cellular degranulation and the immediate release of various preformed (e.g. histamine, tryptase, carboxypeptidase A, and proteoglycans), or de novo synthesized (e.g. prostaglandins, leukotrienes, platelet-activating factor and cytokines) soluble mediators.
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; 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.
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: Human IgE, Human FcεRIα (huIgE/huFcεRIα)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Human IgE, Human FcεRIα (huIgE/huFcεRIα)
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