PSA fluoroimmunoassays using anti-PSA ScFv and quantum-dot conjugates

Area of Science:

• Bioconjugation Chemistry
• Nanoparticle-Antibody Interactions
• Biomolecular Engineering

Background:

• Monoclonal antibody-nanoparticle conjugates are vital for fluoroimmunoassays and bioimaging.
• Direct conjugation of full-length antibodies to nanoparticles often impairs binding activity due to aggregation.
• Single-chain antibody fragments (scFvs) offer advantages in solubility, activity, and engineering.

Purpose of the Study:

• To evaluate the conjugation of engineered scFvs with quantum dots (Qdots).
• To compare the binding activities of different scFv-Qdot conjugates.
• To demonstrate the impact of structure-based genetic engineering on scFv/Qdot conjugate performance.

Main Methods:

• Preparation of two antiprostate-specific antigen scFv mutants (scFvB80-M1 and scFvB80-M2) with distinct C-terminal tags.
• Covalent conjugation of scFv mutants directly with Cadmium Selenide/Zinc Sulfide (CdSe/ZnS) Qdots.
• Measurement and comparison of binding activities of the resulting scFv/Qdot conjugates in fluoroimmunoassays.

Main Results:

• Both scFv mutants were successfully conjugated to CdSe/ZnS Qdots.
• The scFvB80-M2/Qdot conjugate exhibited binding activity equivalent to free scFvB80.
• The scFvB80-M1/Qdot conjugate showed four times lower binding activity compared to scFvB80-M2/Qdots.

Conclusions:

• Covalent conjugation of scFvs to Qdots is feasible.
• Structure-based genetic engineering of scFvs significantly impacts the binding affinity of scFv/Qdot conjugates.
• Optimized scFv engineering can enhance the utility of nanoparticle-based diagnostic and imaging tools.