Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates.

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

The endosomal proteome of macrophage and dendritic cells.

The essential roles of the endovacuolar system in health and disease call for the development of new tools allowing a better understanding of the complex molecular machinery involved in endocytic processes. We took advantage of the floating properties of small latex beads (sLB) on a discontinuous sucrose gradient to isolate highly purified endosomes following internalization of small latex beads in J774 macrophages and bone marrow-derived dendritic cells (DC). We particularly focused on the isolation of macrophages early endosomes and late endosomes/lysosomes (LE/LYS) as well as the isolation of LE/LYS from immature and lipopolysaccharide-activated (mature) DC. We subsequently performed a comparative analysis of their respective protein contents by MS. As expected, proteins already known to localize to the early endosomes were enriched in the earliest fraction of J774 endosomes, while proteins known to accumulate later in the process, such as hydrolases, were significantly enriched in the LE/LYS preparations. We next compared the LE/LYS protein contents of immature DC and mature DC, which are known to undergo massive reorganization leading to potent immune activation. The differences between the protein contents of endocytic organelles from macrophages and DC were underlined by focusing on previously poorly characterized biochemical pathways, which could have an unexpected but important role in the endosomal functions of these highly relevant immune cell types.

Advanced procedures for labeling of antibodies with quantum dots.

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.

Semiconductor quantum dots for multiplexed bio-detection on solid-state microarrays.

Understanding cellular systems requires identification and analysis of their multiple components and determination of how they act together and are regulated. Microarray technology is one of the few tools that is able to solve such problems. It is based on high-throughput recognition of a target to the probe and has the potential to simultaneously measure the presence of numerous molecules in multiplexed tests, all contained in a small drop of test fluid. Microarrays allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signal read-out from the microarrays is done with organic dyes which often suffer of photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals named "quantum dots" (QDs) allows to push these limits away. QDs are sufficiently bright to be detected as individual particles, extremely resistant against photobleaching and provide unique possibilities for multiplexing, thus supplying the microarray technology with a novel read-out option enabling the sensitivity of detection to reach the single-molecule level. This paper reviews QDs applications to microarray-based detection and demonstrates how the combination of microarray and QDs technologies may increase sensitivity and highly parallel capacities of multiplexed microarrays. Such a combination should provide the breakthrough results in drug discovery, cancer diagnosis and establish new therapeutic approaches through the identification of binding target molecules and better understanding of cell signalling pathways.