Generation of Thermally Stable Affinity Pairs for Sensitive, Specific Immunoassays

Area of Science:

• Biotechnology
• Protein Engineering
• Diagnostic Development

Background:

• Point-of-care diagnostic tests often utilize pairs of monoclonal antibodies to detect specific molecules.
• Generating and validating these antibody pairs, including epitope binning, can be time-consuming.
• There is a need for alternative, rapidly generated affinity reagents for diagnostics.

Purpose of the Study:

• To describe a protocol for identifying and generating affinity pairs using a small protein scaffold, rcSso7d, as a substitute for monoclonal antibodies.
• To demonstrate that this approach can accelerate the development of diagnostic tools.
• To enable the rapid synthesis of affinity pairs in standard bacterial expression systems.

Main Methods:

• Utilizing a large yeast display library to screen for high-affinity binding variants of the rcSso7d protein scaffold.
• Identifying specific protein variants that bind to distinct epitopes of a target molecule.
• Synthesizing the selected affinity pairs using common bacterial protein expression strains.

Main Results:

• Successfully identified strong binding variants of the rcSso7d scaffold capable of forming affinity pairs.
• Demonstrated that this method is significantly faster than traditional antibody generation and epitope binning.
• The generated affinity pairs can be synthesized efficiently in bacterial systems.

Conclusions:

• The rcSso7d protein scaffold offers a viable and rapid alternative to monoclonal antibodies for creating diagnostic affinity pairs.
• This protocol facilitates the accelerated development and production of novel point-of-care diagnostic tools.
• The described method streamlines reagent generation, potentially reducing costs and time-to-market for new diagnostics.