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1.
Methods Mol Biol ; 2804: 91-100, 2024.
Article in English | MEDLINE | ID: mdl-38753142

ABSTRACT

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.


Subject(s)
Immunomagnetic Separation , Lab-On-A-Chip Devices , Neoplastic Cells, Circulating , Neoplastic Cells, Circulating/pathology , Immunomagnetic Separation/methods , Humans , Cell Separation/methods , Cell Separation/instrumentation , Neoplasms/pathology , Neoplasms/blood , Cell Line, Tumor , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods
2.
Micromachines (Basel) ; 15(3)2024 Mar 12.
Article in English | MEDLINE | ID: mdl-38542626

ABSTRACT

Given the growing importance of lab-on-a-chip in a number of fields, such as medical diagnosis or environmental analysis, the fact that the current fabrication process relies mainly on oil-based polymers raises an ecological concern. As an eco-responsible alternative, we presented, in this article, a manufacturing process for microfluidic devices from chitosan, a bio-sourced, biodegradable, and biocompatible polysaccharide. From chitosan powder, we produced thick and rigid films. To prevent their dissolution and reduce their swelling when in contact with aqueous solutions, we investigated a film neutralization step and characterized the mechanical and physical properties of the resulting films. On these neutralized chitosan films, we compared two micropatterning methods, i.e., hot embossing and mechanical micro-drilling, based on the resolution of microchannels from 100 µm to 1000 µm wide. Then, chitosan films with micro-drilled channels were bonded using a biocompatible dry photoresist on a glass slide or another neutralized chitosan film. Thanks to this protocol, the first functional chitosan microfluidic devices were prepared. While some steps of the fabrication process remain to be improved, these preliminary results pave the way toward a sustainable fabrication of lab-on-a-chip.

3.
Lab Chip ; 22(21): 4151-4166, 2022 10 25.
Article in English | MEDLINE | ID: mdl-36148526

ABSTRACT

The isolation of circulating tumor cells (CTCs) directly from blood, as a liquid biopsy, could lead to a paradigm shift in cancer clinical care by providing an earlier diagnosis, a more accurate prognosis, and personalized treatment. Nevertheless, CTC-specific challenges, including their rarity and heterogeneity, have hampered the wider use of CTCs in clinical studies. Microfluidic-based isolation technologies have emerged as promising tools to circumvent these limitations but still fail to meet the constraints of high purity and short processing time required to ensure compatibility with clinical follow-up. In this study, we developed an immunomagnetic-based microfluidic device, the MagPure chip, to achieve the negative selection of CTCs through the depletion of white blood cells (WBCs) and provide highly purified samples for subsequent analysis. We demonstrate that the MagPure chip depletes all magnetically labeled WBCs (85% of WBCs were successfully labeled) and ensures a CTC recovery rate of 81%. In addition, we show its compatibility with conventional biological studies, including 2D and 3D cell culture, as well as phenotypic and genotypic analyses. Finally, we successfully implemented a two-step separation workflow for whole blood processing by combining a size-based pre-enrichment system (ClearCell FX1®) with the MagPure chip as a subsequent purification step. The total workflow led to high throughput (7.5 mL blood in less than 4 h) and high purity (947 WBCs per mL remaining, 99.99% depletion rate), thus enabling us to quantify CTC heterogeneity in size and tumor marker expression level. This tumor-marker-free liquid biopsy workflow could be used in a clinical context to assess phenotype aggressiveness and the prognosis rate.


Subject(s)
Microfluidic Analytical Techniques , Neoplastic Cells, Circulating , Humans , Neoplastic Cells, Circulating/pathology , Lab-On-A-Chip Devices , Cell Separation , Cell Line, Tumor , Liquid Biopsy , Biomarkers, Tumor
4.
Biosens Bioelectron ; 197: 113770, 2022 Feb 01.
Article in English | MEDLINE | ID: mdl-34768065

ABSTRACT

Cancer is one of the leading cause of death worldwide. Lung cancer (LCa) and prostate cancer (PCa) are the two most common ones particularly among men with about 20% of aggressive metastatic form leading to shorter overall survival. In recent years, circulating tumor cells (CTCs) have been investigated extensively for their role in metastatic progression and their involvement in reduced overall survival and treatment responses. Analysis of these cells and their associated biomarkers as "liquid biopsy" can provide valuable real-time information regarding the disease state and can be a potential avenue for early-stage detection and possible selection of personalized treatments. This review focuses on the role of CTCs and their associated biomarkers in lung and prostate cancer, as well as the shortcomings of conventional methods for their isolation and analysis. To overcome these drawbacks, biosensors are an elegant alternative because they are capable of providing valuable multiplexed information in real-time and analyzing biomarkers at lower concentrations. A comparative analysis of different transducing elements specific for the analysis of cancer cell and cancer biomarkers have been compiled in this review.


Subject(s)
Biosensing Techniques , Neoplastic Cells, Circulating , Prostatic Neoplasms , Biomarkers, Tumor , Humans , Lung , Male , Prostatic Neoplasms/diagnosis
5.
Cells ; 10(7)2021 07 09.
Article in English | MEDLINE | ID: mdl-34359904

ABSTRACT

Magnetophoresis-based microfluidic devices offer simple and reliable manipulation of micro-scale objects and provide a large panel of applications, from selective trapping to high-throughput sorting. However, the fabrication and integration of micro-scale magnets in microsystems involve complex and expensive processes. Here we report on an inexpensive and easy-to-handle fabrication process of micrometer-scale permanent magnets, based on the self-organization of NdFeB particles in a polymer matrix (polydimethylsiloxane, PDMS). A study of the inner structure by X-ray tomography revealed a chain-like organization of the particles leading to an array of hard magnetic microstructures with a mean diameter of 4 µm. The magnetic performance of the self-assembled micro-magnets was first estimated by COMSOL simulations. The micro-magnets were then integrated into a microfluidic device where they act as micro-traps. The magnetic forces exerted by the micro-magnets on superparamagnetic beads were measured by colloidal probe atomic force microscopy (AFM) and in operando in the microfluidic system. Forces as high as several nanonewtons were reached. Adding an external millimeter-sized magnet allowed target magnetization and the interaction range to be increased. Then, the integrated micro-magnets were used to study the magnetophoretic trapping efficiency of magnetic beads, providing efficiencies of 100% at 0.5 mL/h and 75% at 1 mL/h. Finally, the micro-magnets were implemented for cell sorting by performing white blood cell depletion.


Subject(s)
Cell Separation , Immunomagnetic Separation , Lab-On-A-Chip Devices , Magnetics , Polymers/chemistry , Humans , Leukocytes/cytology , Microtechnology , Tomography, X-Ray
6.
Cancer Biomark ; 18(2): 105-116, 2017.
Article in English | MEDLINE | ID: mdl-27983529

ABSTRACT

Thanks to their specificity and stability in the sera, autoantibodies (AAbs) against tumor-associated antigens (TAAs) are very attractive biomarkers for the development of less invasive serological tests for the diagnosis and prognosis of cancer. Heat shock proteins (HSP) belong to TAAs and they are over-expressed in various human cancers. Elevated HSP can stimulate the immune system to produce anti-HSP antibodies. So far, AAbs against HSP have been identified in the circulation of various cancer patients. Here we will review current literature on the use of anti-HSP antibodies for cancer diagnosis and prognosis. The challenges as well as future directions of AAbs identification in oncology are also discussed.


Subject(s)
Autoantibodies/blood , Biomarkers, Tumor/blood , Heat-Shock Proteins/immunology , Neoplasms/diagnosis , Neoplasms/immunology , Antigens, Neoplasm/blood , Biomarkers, Tumor/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Neoplasms/blood
7.
Acta Biomater ; 46: 323-335, 2016 12.
Article in English | MEDLINE | ID: mdl-27686041

ABSTRACT

High-performance bioinert ceramics such as zirconia have been used for biomedical devices since the early seventies. In order to promote osseointegration, the historical solution has been to increase the specific surface of the implant through roughness. Nevertheless these treatments on ceramics may create defects at the surface, exposing the material to higher chances of early failure. In zirconia, such treatments may also affect the stability of the surface. More recently, the interest of improving osseointegration of implants has moved the research focus towards the actual chemistry of the surface. Inspired by this, we have adapted the current knowledge and techniques of silica functionalization and applied it to successfully introduce 3-aminopropyldimethylethoxy silane (APDMES) directly on the surface of zirconia (3Y-TZP). We used plasma of oxygen to clean the surface and promote hydroxylation of the surface to increase silane density. The samples were extensively characterized by means of X-ray photoelectron spectroscopy (XPS) and contact angle, mechanically tested and its cytotoxicity was evaluated through cell adhesion and proliferation tests. Additionally, aging was studied to discard negative effects of the treatment on the stability of the tetragonal phase. No adverse effect was found on the mechanical response of treated samples. In addition, plasma-treated samples exhibited an unexpectedly higher resistance to aging. Finally, silane density was 35% lower than the one reported in literature for silica. However cells displayed a qualitatively higher spreading in opposition to the rounder appearance of cells on untreated zirconia. These results lay the foundations for the next generation of zirconia implants with biologically friendlier surfaces. STATEMENT OF SIGNIFICANCE: The use of zirconia-based ceramics in biomedical devices is broad and well accepted, especially in dental implants. However, they do not bond naturally to bone, therefore to ensure fixation surgeons typically rely on roughness at different scales, or on cements. Alternatively in this work we present a new perspective of surface modification through chemistry to enhance the interaction between surface and biological environment, without the downsides of roughness. This surface treatment is proposed for zirconia, which allowed a direct silanization of its surface and a higher cell attachment. The results of this research may open the possibility for the next generation of bioinert ceramic implants with more advanced tailored surfaces for increased osseointegration.


Subject(s)
Osseointegration/drug effects , Silanes/chemistry , Zirconium/pharmacology , Cell Line , Cell Proliferation/drug effects , Humans , Kinetics , Photoelectron Spectroscopy , Surface Properties
8.
Anal Bioanal Chem ; 408(5): 1497-506, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26715250

ABSTRACT

Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%.


Subject(s)
Autoantibodies/blood , Biomarkers, Tumor/blood , Breast Neoplasms/diagnosis , Heat-Shock Proteins/immunology , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Antigens, Neoplasm/blood , Autoantibodies/immunology , Biomarkers, Tumor/immunology , Breast Neoplasms/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoassay/methods , Neoplasm Staging , Prospective Studies
9.
Curr Opin Chem Biol ; 18: 46-54, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24440778

ABSTRACT

The present review concerns the recent advances in DNA directed immobilization (DDI) based glycocluster array. The impact of glycan immobilization on subsequent interactions with protein is discussed and the consequent pros and cons of DDI-based glycocluster array are reviewed. Finally, application in the discovery of anti-pathogen molecules is illustrated by screening for galactose or fucose glycoclusters targeting two Pseudomonas aeruginosa virulence factors (PA-IL and PA-IIL).


Subject(s)
DNA/metabolism , Fucose/analysis , Galactose/analysis , Glycomics/methods , Fucose/metabolism , Galactose/metabolism , Pseudomonas aeruginosa/metabolism , Virulence Factors/analysis , Virulence Factors/metabolism
10.
Langmuir ; 29(5): 1498-509, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23305497

ABSTRACT

Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.


Subject(s)
Amines/chemistry , Antibodies/chemistry , Biomarkers, Tumor/analysis , Chitosan/chemistry , Colorectal Neoplasms/diagnosis , Propylamines/chemistry , Silanes/chemistry , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Protein Array Analysis , Surface Properties
11.
Biosens Bioelectron ; 40(1): 385-92, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23017679

ABSTRACT

There is an urgent need to identify relevant tumor markers showing high sensitivity and specificity for early diagnosis and prognosis of breast cancer. Protein microarrays have demonstrated to be cost-effective, high through-put and powerful tools for screening and identifying tumor markers with only minute samples. Autoantibodies directed against tumor-associated antigens (TAAs) were shown to be relevant tumor markers. However, due to the variability of immune response from one individual to another and depending on the type of cancer, detection of only one type of anti-TAA autoantibody is not sufficient to give a reliable and precise diagnosis. It is necessary to use a set of several TAAs for determining specific autoimmune profiles. Therefore, combining various TAAs on different surfaces could improve sensitivity and specificity for anti-TAA autoantibody detection. Herein a panel of 10 proteins, including well-known tumor-associated antigens (TAAs) and potential new biomarkers of breast cancer, were immobilized onto microstructured microarray under optimized conditions (spotting pH buffer, surface chemistry, blocking procedure), in order to determine an autoimmune signature of breast cancer. Sera from 29 breast cancer patients and 28 healthy donors were screened in sandwich immunoassays on the miniaturized system to detect the eventual presence of anti-TAAs autoantibodies. Results indicated that the detection level of each anti-TAA autoantibody in a given serum sample was strongly dependant on the surface chemistry. Combining five TAAs (p53, Hsp60, Hsp70, Her2-Fc, NY-ESO-1) on two different surface chemistries (NHS and APDMES) allowed the significant detection of more than 82% breast cancer sera.


Subject(s)
Antigens, Neoplasm/blood , Biomarkers, Tumor/blood , Biosensing Techniques/instrumentation , Breast Neoplasms/blood , Immunoassay/instrumentation , Protein Array Analysis/instrumentation , Breast Neoplasms/diagnosis , Equipment Design , Equipment Failure Analysis , Female , Humans , Protein Binding , Reproducibility of Results , Sensitivity and Specificity
12.
Biosens Bioelectron ; 26(4): 1320-5, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-20678917

ABSTRACT

Peptide microarray can be implemented by immobilization of peptides on a solid support or by direct on-chip peptide synthesis (OCPS). In the first case, peptide primary sequences can be ensured prior to their immobilization but structural diversity is achieved at high cost in terms of reagents. In the second case, high diversity is achieved with low amount of reagents but the primary and secondary structures cannot be ascertained. In both cases, the immobilization step will influence the overall biological activity. We proposed a strategy where direct peptide on-chip synthesis and peptide immobilization are viewed as complementary approaches. In a first step, OCPS is envisioned for the screening and selection of biologically relevant peptides. In a second step, selected peptides would be synthesized on resin, qualified and immobilized for implementing microarrays (i.e. for diagnosis). A versatile surface chemistry for both OCPS and peptide immobilization was developed allowing for an identical physico-chemical environment for both implementation strategies. In the present report, a 16 mer peptide corresponding to the human histone H3 epitope was synthesized on an amino-functionalized support. Surface stability (including upon deprotection steps) and peptide primary and secondary structures were assessed with Cy3-streptavidine conjugates and with immunoassays. Peptides, either on-chip synthesized or immobilized, exhibited a similar biological activity.


Subject(s)
Antibodies/analysis , Histones/immunology , Protein Array Analysis/methods , Amino Acid Sequence , Animals , Epitopes/chemistry , Epitopes/genetics , Fluorescent Dyes , Histones/chemistry , Histones/genetics , Humans , Immunoassay/instrumentation , Immunoassay/methods , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/immunology , Protein Array Analysis/instrumentation , Rabbits , Surface Properties
13.
Anal Biochem ; 400(1): 10-8, 2010 May 01.
Article in English | MEDLINE | ID: mdl-20079705

ABSTRACT

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.


Subject(s)
Immunoassay/methods , Protein Array Analysis/methods , Autoantibodies/blood , Blotting, Western/methods , Enzyme-Linked Immunosorbent Assay/methods , Histones/chemistry , Histones/immunology , Histones/metabolism , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/immunology , Immobilized Proteins/metabolism , Lupus Erythematosus, Systemic/metabolism , Miniaturization , Pyran Copolymer/chemistry , Silanes/chemistry
14.
Article in English | MEDLINE | ID: mdl-18002437

ABSTRACT

Protein microarray technology has shown great advancements in the field of biomedical research and diagnosis, it allows to study and understand protein activities and protein - ligand interactions (e.g. detection of antigen-autoantibody interaction in autoimmune diseases. Autoantibodies are frequently targeted against antigens of the cell nucleus (double and single stranded DNA, histones, and nuclear antigens). The biological activities of proteins (e.g. enzymes, antibodies...) are controlled by peptides sequences of the active site. Consequently, we were interested in the investigation of peptide microarrays in order to further implement in situ peptide synthesis, in particular, deprotection reaction on glass supported peptides. In this work, a protected and biotinylated synthetic peptide was covalently immobilized onto amino functionalized glass surface by activation of its the C-terminus; this allows to orientate the peptide onto the surface. The peptide contains a fragment of the C-terminal end of the human histone H3 protein. The immobilized peptide was then deprotected by using concentrated trifluoroacetic acid solution. After the deprotection, surface stability and peptide grafting density were evaluated by indirect labelling of the immobilized peptide using Cy3 streptavidin conjugates. We also studied biological interaction of IgG polyclonal anti-histone H3 antibody with the immobilized peptide epitope to insure the efficiency of the acid deprotection. The specificity of the antibody interaction with the protected versus non protected peptides. This approach may be applied to in situ synthetic and prototected peptides, in order to elaborate a micro-immunoassay prototype for measurement of peptide-protein interactions on high density microarrays, and detection of antibodies in biological fluids such as serum.


Subject(s)
Glass , Histones/chemistry , Immunoglobulin G/chemistry , Peptide Library , Peptides/chemistry , Protein Array Analysis/instrumentation , Proteins/chemistry , Amino Acid Sequence , Antibodies/chemistry , Biotinylation , Enzyme-Linked Immunosorbent Assay/methods , Equipment Design , Humans , Ligands , Molecular Sequence Data
15.
Anal Biochem ; 340(1): 14-23, 2005 May 01.
Article in English | MEDLINE | ID: mdl-15802125

ABSTRACT

A serological chemiluminescent biochip was designed based on screen-printed electrode arrays composed of nine 1-mm(2) electrodes. Arrays were shown to be produced with good batch-to-batch reproducibility (standard deviations of 4.4 and 12.0% for ferricyanide oxidation potential and current, respectively) and very good reproducibility within a particular array (2.0 and 7.5% standard deviations for the same controls). Electrode arrays were used to electroaddress various bioconjugate structures comprising a recombinant HIV-1 P24 capsid protein (RH24K) in polypyrrole film. Entrapment of RH24K preimmobilized onto maleic anhydride-alt-methyl vinyl ether copolymer was shown to be the more efficient immobilization procedure. This addressed sensing layer enabled the detection of anti-P24 antibodies at a concentration of 3.5 ng/ml through peroxidase-labeled anti-human immunoglobulin G reaction. The biochip was used to perform an HIV-1 serological test in human sera. HIV-1 seropositive and seronegative sera were easily discriminated using serum dilutions greater than 1/10,000.


Subject(s)
HIV Antibodies/analysis , HIV Antibodies/immunology , HIV Core Protein p24/chemistry , HIV Core Protein p24/immunology , Serologic Tests/instrumentation , Serologic Tests/methods , Amino Acid Sequence , Electrodes , HIV Seronegativity/immunology , HIV Seropositivity/immunology , Humans , Immune Sera/immunology , Kinetics , Molecular Sequence Data , Molecular Structure , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Reproducibility of Results , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
16.
Anal Bioanal Chem ; 381(5): 1019-24, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15599499

ABSTRACT

Two diagnostic chemiluminescent biochips were developed for either the detection of p53 gene point mutation or the serological detection of anti-HIV-1 p24 capsid protein. Both biochips were composed of 24 microarrays of latex beads spots (4x4) (150 microm in diameter, 800 microm spacing) entrapped in a poly(dimethylsiloxane) elastomer (PDMS). The latex beads, bearing oligonucleotide sequences or capsid protein, were spotted with a conventional piezoelectric spotter and subsequently transferred at the PDMS interface. The electron microscopy observation of the biochips showed how homogeneous and well distributed the spots could be. Point mutation detection on the codon 273 of the p53 gene was performed on the basis of the melting temperature difference between the perfect match sequence and the one base pair mismatch sequence. The hybridisation of a 20-mer oligonucleotide form the codon 273 including a one base pair mutation in its sequence on a biochip arrayed with non-muted and the muted complementary sequences, enabled a clear discrimination at 56 degrees C between muted and wild sequences. Moreover, the quantitative measurement of the amount of muted sequence in a sample was possible in the range 0.4-4 pmol. Serological measurement of anti-HIV-1 p24 capsid protein on the biochip, prepared with 1-microm-diameter latex beads, enabled the detection of monoclonal antibodies in the range 1.55-775 ng mL(-1). Such a range could be lowered to 0.775 ng mL(-1) when using 50-nm-diameter beads, which generated a higher specific surface. The validation of the biochip for the detection of anti-HIV-1 capsid protein antibodies was performed in human sera from seropositive and seronegative patients. The positivity of the sera was easily discriminated at serum dilutions below 1:1,000.


Subject(s)
Capsid Proteins/immunology , Dimethylpolysiloxanes/chemistry , Genes, p53 , HIV Antibodies/blood , HIV-1/immunology , Microarray Analysis/methods , Nylons/chemistry , Point Mutation , Capsid Proteins/chemistry , Cells, Immobilized/chemistry , Humans , Luminescent Measurements , Microspheres
17.
Bioconjug Chem ; 15(3): 458-66, 2004.
Article in English | MEDLINE | ID: mdl-15149172

ABSTRACT

We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavière, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.


Subject(s)
Anhydrides/chemistry , HIV Core Protein p24 , Polymers/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Amino Acid Sequence , Epitopes , HIV Core Protein p24/chemistry , HIV Core Protein p24/immunology , Immobilized Proteins , Lysine/chemistry , Molecular Sequence Data , Peptide Mapping , Recombinant Proteins/isolation & purification , Stereoisomerism
18.
J Med Virol ; 69(4): 503-9, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12601758

ABSTRACT

Outbreaks of new or old diseases appear primarily in tropical zones such as Africa, south and central America, or Asia. Among these diseases, those induced by Arboviruses (the best known of which are being yellow fever, dengue, Ebola, and Sindbis) are under intensive observation by the World Health Organization. Rapid isolation and identification of the viral species is the first step in the diagnosis, study, and control of epidemics. One major problem with the isolation of viruses is capturing sufficient numbers of viral particles to test. The work presented in this report addresses this question. We have tested the interaction between Babanki (Togaviridae), Kedougou (Flaviviridae) viruses, and a range of insoluble polystyrene derivatives substituted with arginine groups. Insoluble functionalized copolymers were found to develop specific interactions with viruses through chemical groups present on their surfaces. The adsorption of viruses varied according to the percentage of arginine substituted onto the polymer, with a maximum value for both viruses of about 20% of grafting rate. It was also found that the Kedougou virus displayed the highest affinity for this polymer.


Subject(s)
Flavivirus/metabolism , Polystyrenes , Sindbis Virus/metabolism , Adsorption , Animals , Arginine , Chlorocebus aethiops , Humans , Microscopy, Electron, Scanning , Microspheres , Polymers/chemical synthesis , Polystyrenes/chemistry , Tritium , Uridine/metabolism , Vero Cells
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