Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats

The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.     

Phage-based molecular directed evolution yields multiple tandem human IgA affibodies with intramolecular binding avidity

Affibodies are a group of affinity proteins that are based on a 58-amino-acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. A single human IgA affibody with high IgA affinity has been generated by directed evolution. It remains interesting whether tandem IgA affibody proteins could increase binding capacity. Here, we report the generation of multiple tandem IgA affibodies by directed evolution using a combinatorial phage library displaying the IgA affibody A1 and/or A2 linked with three random amino acids. These affibodies exhibited markedly increased IgA binding capacity, as shown by enzyme linked immunosorbent assay, immunoblotting and surface plasmon resonance assays. We further showed that these tandem IgA affibodies displayed preferential binding to intact IgA molecules compared to individual IgA chain, suggesting intramolecular binding avidity. Our data demonstrates that artificial multiple tandem human IgA affibodies with relevant biological binding avidity were successfully yielded by phage-based molecular evolution. These results have broad implications for the design and development of binding proteins that target important biological molecules.     

Structural basis for molecular recognition in an affibody:affibody complex

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.     

Thermodynamics of folding and binding in an affibody:affibody complex

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.     

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A.

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (K(d)) in the range between 0.5 and 3 microm. One variant (Z(IgA1)) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA(1) and IgA(2)) as well as secretory IgA were recognized with similar efficiencies. No detectable cross-reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the Z(IgA1) affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head-to-tail Z(IgA1) affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.     

Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs

A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.     

Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.     

Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of (111)In-Labeled Synthetic Anti-HER2 Affibody Molecules

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.     

Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ?-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((??m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.     

Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering

CD28 is one of the key molecules for co-stimulatory signalling in T cells. Here, novel ligands (affibodies) showing selective binding to human CD28 (hCD28) have been selected by phage display technology from a protein library constructed through combinatorial mutagenesis of a 58-residue three-helix bundle domain derived from staphylococcal protein A. Analysis of selected affibodies showed a marked sequence homology and biosensor analyses showed that all investigated affibodies bound to hCD28 with micromolar affinities (KD). No cross-reactivity towards the related protein human CTLA-4 could be observed. This lack of cross-reactivity to hCTLA-4 suggests that the recognition site on hCD28 for the affibodies resides outside the conserved MYPPPYY motif. The apparent binding affinity for hCD28 could be improved through fusion to an Fc fragment fusion partner, resulting in a divalent presentation of the affibody ligand. For the majority of selected anti-CD28 affibodies, in co-culture experiments involving Jurkat T-cells and CHO cell lines transfected to express human CD80 (hCD80) or LFA-3 (hLFA-3) on the cell surface, respectively, pre-incubation of Jurkat cells with affibodies resulted in inhibition of IL-2 production when they were co-cultured with CHO (hCD80+) cells, but not with CHO (hLFA-3+) cells. For one affibody variant denoted Z(CD28:5) a clear concentration-dependent inhibition was seen, indicating that this affibody binds hCD28 and specifically interferes in the interaction between hCD28 and hCD80.     

Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K(D)) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both K(D) and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.     

A metal-chelating piezoelectric sensor chip for direct detection and oriented immobilization of polyHis-tagged proteins

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resulting chip effectively captured Ni2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. The real-time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long-term reusability and storage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealed that Co2+, Cu2+, and Ni2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni2+-charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni2+-charged chip and of its substrate with a protein-coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis-tagged protein and its functional study using a real-time PZ biosensor.     

Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with flow cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K_D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both K_D and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.     

Photonic immobilization of BSA for nanobiomedical applications: creation of high density microarrays and superparamagnetic bioconjugates

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe(3)O(4)@Au core-shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system.     

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ~100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.