Anti-Fc capture system: convenient and fast immobilization of antibodies

The anti-Fc capture system immobilizes antibodies on an oligonucleotide sensor chip through oligo-conjugated Protein A/G or Protein A/G/L. This application note tests both proteins against antibody types and subclasses from several species.

The Challenge: Immobilizing Antibodies Without Changing Them

Labeling or covalent coupling takes time and can affect antibody function. A covalently bound antibody can also be hard to regenerate. A capture layer avoids these steps, but it must hold many antibody species and subclasses for long enough to measure.

The Approach: Capture, Dissociation and Regeneration Cycles

Protein A/G-oligo or Protein A/G/L-oligo (50 nM) was hybridized to the sensor chip on a MACS Matchmaker. IgG (500 nM) was captured for 10 minutes, followed by 10 minutes of dissociation at 100 µL/min in PBS-T. Each cycle ended with regeneration, which removed the capture protein and the IgG, and the sensor chip was reused.

Key Results Across Antibody Species and Subclasses

• Protein A/G: active toward all tested antibodies except mouse IgM, hamster IgG lambda and goat IgG.
• Protein A/G/L: captured hamster IgG lambda and goat IgG, but not two mouse IgM subclasses, mouse IgG1 lambda or rat IgG2a lambda.
• Kappa chains: the added L domain did not change activity or off-rates much for kappa IgGs.
• Estimated k_off: most captured IgGs fell in the 10⁻⁴ s⁻¹ range, measured over 10 minutes.

Why It Matters for Antibody Screening

Neither protein offers a general advantage, except in specific cases shown in the note. Both suit a fast first screening, not a final characterization, because capture can alter the antibody–antigen interaction. Only analytes with a k_off greater than that of the captured IgG can be examined.