Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

Alyona Sukhanova M.D.; Klervi Even-Desrumeaux; Jean-Marc Millot; Patrick Chames; Daniel Baty; Mikhail Artemyev; Vladimir Oleinikov; Jacques H. M. Cohen M.D.; Igor Nabiev

doi:10.1117/12.905896

2 February 2012 Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

Alyona Sukhanova M.D., Klervi Even-Desrumeaux, Jean-Marc Millot, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov, Jacques H. M. Cohen M.D., Igor Nabiev

Author Affiliations +

Proceedings Volume 8232, Colloidal Nanocrystals for Biomedical Applications VII; 82320T (2012) https://doi.org/10.1117/12.905896
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

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Alyona Sukhanova M.D., Klervi Even-Desrumeaux, Jean-Marc Millot, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov, Jacques H. M. Cohen M.D., and Igor Nabiev "Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications", Proc. SPIE 8232, Colloidal Nanocrystals for Biomedical Applications VII, 82320T (2 February 2012); https://doi.org/10.1117/12.905896

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