Linking Single Domain Antibodies that Recognize Different Epitopes on the Same Target.

Area of Science:

• Biotechnology
• Immunology
• Biochemistry

Background:

• Single domain antibodies (sdAbs) are derived from heavy-chain-only antibodies in camelids and sharks.
• SdAbs exhibit high affinity antigen binding and can refold after denaturation.
• Previous work isolated ricin-binding sdAbs targeting four distinct epitopes.

Purpose of the Study:

• To construct and evaluate genetically linked sdAb pairs for enhanced ricin detection.
• To investigate the impact of linker length and orientation on sdAb pair functionality.
• To compare the performance of linked sdAb pairs against individual sdAbs and monoclonal antibodies.

Main Methods:

• Genetic linkage of sdAb pairs using peptide linkers of varying lengths.
• Construction of linked sdAb pairs in both possible orientations.
• Assessment of sdAb binding to ricin and simultaneous binding capability.
• Evaluation of refolding ability after denaturation.

Main Results:

• Genetically linked sdAb pairs demonstrated simultaneous binding to ricin.
• The order of sdAbs within the linked pair significantly influenced performance.
• Linked sdAb pairs achieved improved ricin detection limits compared to monoclonal antibodies.
• Refolding capacity of linked sdAbs was reduced compared to unlinked sdAbs.

Conclusions:

• The sequential arrangement of sdAbs in genetically linked pairs is crucial for optimal antigen binding.
• Linked sdAbs offer a promising strategy for sensitive ricin detection, despite some compromise in refolding ability.