Single-domain protein A-engineered magnetic nanoparticles: toward a universal strategy to site-specific labeling of antibodies for targeted detection of tumor cells.
Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T(2) contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor.
Mazzucchelli
Serena
S
Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Ospedale L. Sacco, via G.B. Grassi 74, 20157 Milano, Italy.
Colombo
Miriam
M
De Palma
Clara
C
Salvadè
Agnese
A
Verderio
Paolo
P
Coghi
Maria D
MD
Clementi
Emilio
E
Tortora
Paolo
P
Corsi
Fabio
F
Prosperi
Davide
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000473:pathology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008279:Magnetic Resonance Imaging / Q000379:methods; D008422:Materials Testing; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D011817:Rabbits; D000068878:Trastuzumab
D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D007074:Immunoglobulin G; D009930:Organic Chemicals; D000068878:Trastuzumab