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

Aims: The conjugates of monoclonal antibodies and luminescent nanoparticles (quantum dots [Qdots]) have a large number of potential applications in both fluoroimmunoassays and biological imaging; however, conjugating full-length antibody monoclonal antibodies directly to Qdots or other inorganic nanoparticles often results in the irreversible formation of oligomeric monoclonal anti body-nanoparticle complexes, which leads to dramatically reduced binding activities. This study demonstrated that the use of single-chain antibody fragments (scFvs) appears to have a number of advantages, in terms of solubility, activity, ease of preparation and ease of structure-based genetic engineering. Materials & methods: Two antiprostate-specific antigen scFvs mutants - one with an 11-residue c-myc (referred as scFvB80-M1) and the other with a lysine-enriched His 6-tagging peptide attached to their C-termini (referred as scFvB80-M2) - were prepared. These two scFv mutants were conjugated directly with CdSe/ZnS Qdots and their binding activities were measured and compared. Results & discussion: Both scFv mutants can be conjugated covalently with CdSe/ZnS Qdots; however, the resulting conjugates exhibit significantly different affinities in the prostate-specific antigen fluoroimmunoassays - the binding activity of scFvB80-M2/Qdots is equivalent of that of free scFvB80 and four times of that of scFvB80-M1/Qdots. Conclusion: This study demonstrates that binding activity of scFv/Qdot conjugates can be improved through structure-based genetic engineering of the scFv.