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Analysis Biomolecular Interactions of Antibody with Various P[8] Molecules by BLI (CAT#: STEM-MB-0116-CJ)

Introduction

Vaccination is a cornerstone of public health for preventing illness and death due to common infectious diseases. Each NRRV antigen is a recombinant fusion-protein consisting of a truncated version of the rotavirus surface protein VP4 (known as VP8), genetically fused to the tetanus toxoid universal CD4+ T-cell epitope (P2). The three fusion-protein antigens derive from the VP8 component of three different RV serotypes, P[8], P[6], and P[4]. These sequence variants of P[8] and P[4] NRRV can be as a case study to demonstrate proof-of-concept for a two-stage formulation developability assessment workflow to rapidly assess formulation variables for recombinant vaccine antigens that could be applied to accelerate development of low-cost vaccine dosage forms targeted for use in low and middle income countries (LMICs).




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; Virology

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: Creoptix® Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
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