Jean-Marc Millot

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes

UNLABELLED Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots

UNLABELLED An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. FROM THE CLINICAL EDITOR This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

Extracellular matrix proteins protect human HT1080 cells against the antimigratory effect of doxorubicin

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two‐dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug‐dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment‐mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs. (Cancer Sci 2008; 99: 1699–1705)

Serum albumin-alginate coated microspheres: role of the inner gel in binding and release of the KRFK peptide.

In continuation with our previous study using fluorescein-isothiocyanate (FITC)-Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4x10(5) L/mol or 8x10(5) L/mol for gelation with 5% or 20% CaCl(2), respectively). Binding site number doubled (from 2.6x10(-7) mol/mg to more than 5x10(-7) mol/mg) when microsphere size decreased from 450 microm to 100 microm. Binding sites were assumed to be located in the gel underneath the membrane.

Association between host species choice and morphological characters of main sensory structures of Culicoides in the Palaeartic region

Culicoides (Diptera: Ceratopogonidae) serve as vectors of several mammalian and avian diseases, including bluetongue, Schmallenberg, African horse sickness, avian malaria and Oropouche. Host preference investigations are necessary to assess the transmission routes of vector-borne diseases and to inform mitigation strategies. A recent study examining the main sensory structures (palps and antennae) of Culicoides species suggests that they be classified as ornithophilic or mammalophilic according to their feeding habits. We analyzed Culicoides host preferences according to the literature and carried out a multiple correspondence analysis linking these preferences with morphological data. Seven out of 12 variables were found to be reliable predictors of host preference in Culicoides species: Antenna Flagellomer-Sensilla Coeloconica-Number: (7–10) and (11–13); Antenna Flagellomer-Sensilla Coeloconica IV–X: presence; Palpus-size: wide and/or narrow opening and shallow pit; Palpus-Shape: strongly swollen; Antenna-Short sensilla trichodea-distal part segment IV to X-Number: 2 seta each. Our results demonstrate that the presence of sensilla coeloconica and the maxillary palpus can be used to separate ornithophilic and mammalophilic or ornithophilic/mammalophilic species.

Diagnostic nanoprobes based on the conjugates of quantum dots and single-domain antibodies for cancer biomarkers detection in immunohistochemistry and flow cytometry

Monoclonal antibodies (mAbs) are often too big for the nanoparticle targeting purpose and the conditions used for their conjugation provide nanoprobes with irregular orientation of mAbs on the surface of nanoparticle. Here, we are reviewing our recent publications reporting on ultra-small nanoprobes engineered through oriented conjugation of quantum dots (QDs) with 13-kDa single-domain antibodies (sdAbs) derived from llama and produced in E. coli. Developed nanoprobes with hydrodynamic diameter below 12 nm contain four homogeneously oriented copies of sdAbs on the surface of each QD. They demonstrated excellent specificity and sensitivity for the quantitative detection of rare biomarker-expressing cells using flow cytometry. The higher diffusibility of sdAbs enables immunohistochemical analysis of thick tissues not accessible to mAbs. The data shows that sdAbs-QD conjugates lead to biopsy tissue labelling displaying an equivalent or even better quality than that obtained with gold standard immunohistochemical diagnostics. Developed sdAbs-QD nanoprobes should find numerous applications in multiplexed high-throughput diagnostics and FRET-based detection platforms.

Multiphoton imaging of tumor biomarkers in situ using highly oriented conjugates of single-domain antibodies and quantum dots

High-quality imaging for immunofluorescence diagnosis in medical practice requires a high sensitivity of labeling and discrimination between the fluorescence of immunostaining probes and tissue autofluorescence. Multiphoton microscopy with excitation in the near-infrared spectral region, far from the region of excitation of tissue autofluorescence, enables deep imaging of biological tissues. CdSe/ZnS quantum dots (QDs) conjugated to single-domain antibodies (sdAbs) are efficient diagnostic nanoprobes with diameters <;12 nm and the two-photon absorption cross section exceeding 49,000 GM. Such nanoprobes ensure clear discrimination of tumor areas from normal tissue with the ratio of sdAb-QD emission to the autofluorescence for two-photon excitation >40 times higher than that for one-photon excitation.

Bi-photon imaging and diagnostics using ultra-small diagnostic probes engineered from semiconductor nanocrystals and single-domain antibodies

Semiconductor fluorescent quantum dots (QDs) have just demonstrated their numerous advantages over organic dyes in bioimaging and diagnostics. One of characteristics of QDs is a very large cross section of their twophoton absorption. A common approach to biodetection by means of QDs is to use monoclonal antibodies (mAbs) for targeting. Recently, we have engineered ultrasmall diagnostic nanoprobes (sdAb–QD) based on highly oriented conjugates of QDs with the single-domain antibodies (sdAbs) against cancer biomarkers. With a molecular weight of only 13 kDa (12-fold smaller than full-size mAbs) and extreme stability and capacity to refolding, sdAbs are the smallest functional Ab fragments capable of binding antigens with affinities comparable to those of conventional Abs. Ultrasmall diagnostic sdAb–QD nanoprobes were engineered through oriented conjugation of QDs with sdAbs. This study is the first to demonstrate the possibility of immunohistochemical imaging of colon carcinoma biomarkers with sdAb–QD conjugates by means of two-photon excitation. The optimal excitation conditions for imaging of the markers in clinical samples with sdAb–QD nanoprobes have been determined. The absence of sample autofluorescence significantly improves the sensitivity of biomarker detection with the use of the two-photon excitation diagnostic setup.