Abstract
Ideal nanoparticle-based nanoprobes should contain on their surface homogeneously oriented highly active affinity molecules, e.g., antibodies (Abs), and should not exceed 15 nm in diameter. Direct conjugation of quantum dots (QDs) with Abs through cross-linking of QD amines with the sulfhydryl groups resulting from the reduction of the Ab disulfide bonds is a generally accepted technique. However, this procedure yields conjugates where Abs are oriented irregularly. This decreases the number of functionally active Abs on the nanoparticle surface, because some Ab recognition sites face inwards and cannot interact with the target moieties. Here, we describe an advanced procedure of Ab reduction, affinity purification, and QD-Ab conjugation with optimized critical steps. We have developed a method for partially reducing the Abs yielding highly functional 75 kDa heavy-light chain Ab fragments. Affinity purification of these Ab fragments followed by their tagging with QDs results in QD-Ab conjugates with largely improved functionality compared to those obtained according to the standard procedures. The new approach can be extended to conjugation of any type of Abs with different semiconductor, noble metal, or magnetic nanocrystals.
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Jaiswal JK, Goldman ER, Mattoussi H et al (2004) Use of quantum dots for live cell imaging. Nat Methods 1:73–78
Xing Y, Rao J (2008) Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging. Cancer Biomark 4:307–319
Parak WJ, Gerion D, Pellegrino T et al (2003) Biological applications of colloidal nanocrystals. Nanotechnology 14:R15–R27
Invitrogen. http://probes.invitrogen.com/media/pis/mp19010.pdf
Liu W, Howarth M, Greytak AB et al (2008) Compact biocompatible quantum dots functionalized for cellular imaging. J Am Chem Soc 130:1274–1284
Xing Y, Chaudry Q, Shen C et al (2007) Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat Protoc 2:1152–1165
Zaman MB, Baral TN, Jakubek ZJ et al (2011) Single-domain antibody bioconjugated near-IR quantum dots for targeted cellular imaging of pancreatic cancer. J Nanosci Nanotechnol 11:3757–3763
Zaman MB, Baral TN, Zhang J et al (2009) Single-domain antibody functionalized CdSe/ZnS quantum dots for cellular imaging of cancer cells. J Phys Chem C 113:496–499
Pathak S, Davidson MC, Silva GA (2007) Characterization of the functional binding properties of antibody conjugated quantum dots. Nano Lett 7:1839–1845
Clarke S, Pinaud F, Beutel O et al (2010) Covalent monofunctionalization of peptide-coated quantum dots for single-molecule assays. Nano Lett 10:2147–2154
Nabiev I, Sukhanova A, Artemyev M et al (2008) Colloidal nanoparticles in biotechnology. In: Elissari A (ed) Fluorescent Colloidal Particles as Detection Tools in Biotechnology Systems. Wiley, New York, pp. 133–168
Sukhanova A, Devy J, Venteo L et al (2004) Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells. Anal Biochem 324:60–67
Sukhanova A, Venteo L, Devy J et al (2002) Highly stable fluorescent nanocrystals as a novel class of labels for immunohistochemical analysis of paraffin-embedded tissue sections. Lab Invest 82:1259–1261
Nie S, Xing Y, Kim GJ et al (2007) Nanotechnology applications in cancer. Annu Rev Biomed Eng 9:257–288
Smith AM, Ruan G, Rhyner MN et al (2006) Engineering luminescent quantum dots for in vivo molecular and cellular imaging. Ann Biomed Eng 34:3–14
Zajac A, Song D, Qian W et al (2007) Protein microarrays and quantum dot probes for early cancer detection. Colloids Surf B Biointerfaces 58:309–314
Hermanson G (2008) Bioconjugate techniques, 2nd edn. Academic, New York
Mahmoud W, Rousserie G, Reveil B et al (2011) Advanced procedures for labeling of antibodies with quantum dots. Anal Biochem 416:180–185
Sukhanova A, Even-Desrumeaux K, Kisserli A et al (2012) Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine: NBM 8:516–525
Acknowledgements
This study was partly supported by the Ministry of Education and Science of the Russian Federation (grant 11.G34.31.0050), by the European Commission through the FP7 Cooperation project NAMDIATREAM (grant NMP-2009-4.0-3-246479) and by the programs HYNNOV and Nano’Mat of the Champagne-Ardenne region, the DRRT Champagne-Ardenne, and the FEDER (France). I.N. acknowledges support of the Russian Science Foundation.
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Brazhnik, K., Nabiev, I., Sukhanova, A. (2014). Advanced Procedure for Oriented Conjugation of Full-Size Antibodies with Quantum Dots. In: Fontes, A., Santos, B. (eds) Quantum Dots: Applications in Biology. Methods in Molecular Biology, vol 1199. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1280-3_4
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DOI: https://doi.org/10.1007/978-1-4939-1280-3_4
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