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ForteBio Interactions Newsletter Biosensor photo

January 2009    VOLUME 2    ISSUE 1

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Anti-human IgG (Fc) Capture Biosensors for Immobilization and Kinetic Characterization of Human IgG and Other Fc-containing Proteins

Bettina Heidecker
Product Development Scientist II

The Anti-Human IgG Fc Capture (AHC) biosensor is designed for kinetic characterization of macromolecular interactions between proteins containing an Fc region and a target analyte. This biosensor enables the immobilization of human IgG or other protein containing a human Fc region using a high-affinity anti-human Fc-specific antibody. AHC biosensors were designed to immobilize hIgG from either buffer or cell culture media. Directly pulling hIgG from cell culture media and performing kinetic analyses is a much-simpler workflow than purification and biotinylation. The AHC biosensors can be regenerated through exposure to low pH. This restores the biosensors’ anti-hIgG surface, allowing reuse of the biosensor to perform additional analyses. Regeneration of the surface requires brief exposure to low pH followed by exposure to a neutral solution. Regeneration typically takes one to two minutes, but may vary depending on the hIgG.

A comparison of different immobilization chemistries shows the ease of use for immobilizing hIgG on the Anti-hIgG Fc Capture biosensor.

All four subclasses of human IgG can be captured. A typical assay workflow is diagrammed in Figure 1, illustrating the individual binding and regeneration steps. The runtime binding chart (Figure 2) shows data collected over three regeneration cycles. Surface conditioning and regeneration was performed with 10 mM glycine, pH 1.7.

Figure 3 shows the first binding cycle of a kinetic assay with surface conditioning in step 1, hIgG loading in step 2, analyte association in step 3 and dissociation in step 4. This was followed with regeneration using low pH and three more binding cycles of hIgG and analyte. In the inset all four analyte binding cycles are overlaid together with the corresponding kinetic fits.

The biosensor surface can be regenerated to its original state with low pH. Most hIgG and Fc containing ligands can be eluted from the biosensor surface with 10 mM Glycine, pH 1.7. The cumulative loss of surface capacity is typically less than 20% across 10 regenerations. Table 1 shows the nm-shift of each human IgG loading step and the calculated kinetic rates for each regeneration cycle. The calculated kinetic binding rates are very comparable across all regeneration cycles despite the reduction in hIgG binding capacity.

Applications

The AHC biosensors provide a flexible platform for thorough kinetic profiling of a hIgG-analyte pair or for screening interaction kinetics of hIgG antibodies with relevant analytes. For screening applications, assays can easily be designed to facilitate screening of 40 hIgGs or more against a single analyte in one experiment. The capture surface can be regenerated and re-used at least 10 times, making the AHC biosensor a cost-effective, easy-to-use solution for fast and accurate hIgG kinetic screening.

  • Typical Assay Parameters
  • Sample volume: 200 μL/well (post-dilution)
  • Hydration solution volume: 200 μL/well
  • Flow rate: 1000 rpm
  • Biosensor hydration and sample plate equilibration: 10 minutes

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