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Analysis Biomolecular Interactions of Macaque IgG and simVRC07 IgG by BLI (CAT#: STEM-MB-0160-CJ)

Introduction

Broadly neutralizing antibodies (bnAbs) arise in human immunodeficiency virus type 1 (HIV-1)-infected individuals to various degrees. These bnAbs represent potential components for passive immunization in humans based on the finding that they protect nonhuman primates at physiologically achievable concentrations. Long-term functional analysis of human bnAbs in nonhuman primates can be complicated by macaque immune responses against the human antibody. The AAV8 vectors can encod a simianized version of the bnAb VRC07 with and without transient immunosuppressive treatment in rhesus macaques and the efficacy of vectored bnAb immunoprophylaxis to protect against mucosal SHIV challenge.




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: 293fectin transfection reagent, Profection kit
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