GEN Exclusives

More »

Tutorials

More »
Jul 1, 2009 (Vol. 29, No. 13)

Fluidics-Free Molecular Interactions Analysis

ForteBio’s Dip and Read Method Simplifies Label-Free Assays

  • Click Image To Enlarge +
    Figure 1. Biolayer interferometry

    Molecular interactions play a myriad of important roles in drug discovery and development. From determining mechanism of action to identifying unwanted biological reactions (e.g., monitoring for an immune response to an administered drug), the study of binding events is a critical part of many phases of therapeutic research. 

    Successful analysis of molecular binding kinetics requires the ability to bring two molecules in close proximity under conditions that can overcome diffusion limitations. Much of the commercially available biosensor instrumentation has relied on microfluidics to flow a sample containing the analyte of interest over a surface with the binding partner immobilized. While providing efficient flow, the use of microfluidics limits the sample types that can be used due to the issues of clogging and fouling. In addition, organics and proteins can bind to the large surface area of the microfluidic system contaminating subsequent samples.

    To overcome these disadvantages, a novel dip-and-read instrument platform has been developed. ForteBio’s Octet system uses a fiber optic based biosensor surface that is dipped into the sample of interest. By moving the biosensor surface to the sample, as opposed to moving the sample to the surface, cross contamination from previous samples can be minimized. Samples, buffers, and reagents all remain in the standard 96-well or 384-well microplate throughout the assay while up to 16 biosensors are simultaneously dipped into each reagent in turn.

    To overcome diffusion limitations, the microplate containing the samples is moved in an orbital motion relative to the biosensor. Molecular binding kinetics determined using this orbital flow in the Octet platform agree well with those determined using more complex microfluidics based instrumentation.

    The Octet instruments utilize biolayer interferometry (BLI) to monitor the increase or decrease in optical thickness of molecules on the fiber optic biosensor surface (Figure 1). 

    BLI is an optical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip  and an internal reference layer (Figure 1). Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time. By monitoring the change in molecular thickness, the binding and dissociation of molecules from solution to a surface-immobilized capture molecule can be observed and rates of association and dissociation can be calculated.

  • Macromolecules and Particles

    Proper assay design is critical to the success of any interaction analysis. The molecule to be immobilized onto the surface should be of high purity and activity. The protein can either be immobilized using a direct coupling method or biotinylated and immobilized onto a Streptavidin-containing surface. Direct coupling and biotinylation using NHS reagents both react with the same amine residues on the protein to be immobilized. 

    Since biotinylation can be controlled by optimizing the ratio of biotin-NHS to protein in solution, immobilizing through biotinylation is preferable when dealing with a delicate protein. In addition, using a biotinylation reagent with a long, flexible linker can also help the protein be more accessible for subsequent binding by the analyte. 

    Kinetic analysis of large molecules such as proteins, DNA, or even larger particles such as phage or microspheres can be done using the Octet. The lack of fluidics, along with the fact that the BLI technology is minimally influenced by molecules in solution, allows for a wide range of sample types including lysates, cell culture supernatants, purified, and phage preparations to be used.



Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...
More »

GEN Poll

More » Poll Results »

Stopping Research Fraud

What is the best approach to curbing scientific misconduct and outright fraud?