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.     

Directed immobilization of recognizing elements of biosensor transducers

Data about the directed immobilisation of recognising structures on metal and silicon surfaces of biosensor transducers are analyzed in the review. Attention is paid to such approaches as pretreatment of surface by silanes, thiol compounds as well as to formation of polyelectrolyte-based layers.     

Electrochemical biosensing based on universal affinity biocomposite platforms

Rigid conducting biocomposites are versatile and effective transducing materials for the construction of a wide range of amperometric biosensors such as immunosensors, genosensors and enzymosensors, particularly if the transducer is bulk-modified with universal affinity biomolecules. The strept(avidin)-graphite-epoxy biocomposite could be considered as an universal immobilization platform whereon biotinylated DNAs, oligonucleotides, enzymes or antibodies can be captured by means of the highly affinity (strept)avidin-biotin reaction. Universal affinity biocomposite-based biosensors offer many potential advantages compared to more traditional electrochemical biosensors commonly based on a biologically surface-modified transducer. The integration of many materials into one matrix is their main advantage. As biological bulk-modified materials, the conducting biocomposites act not only as transducers, but also as reservoir for the biomaterial. After its use, the electrode surface can be renewed by a simple polishing procedure, establishing a clear advantage of these approaches relative to classical biosensors and other common biological assays. Moreover, the same material is useful for the analysis of many molecules whose determinations are based on genetic, enzymatic or immunological reactions. The different strategies for electrochemical genosensing, immunosensing and enzymosensing, all of them being dependent on the presence of a redox enzyme marker for the generation of the electrochemical signal, based on this universal affinity biocomposite platform are all presented and discussed.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Photopolymers as an immobilized matrix in biosensing

Biosensors are perspective devices for analysis of substances in chemistry, biological chemistry, medicine, biotechnology and also for environment status monitoring. Their advantages are compact size, short time of analysis, display high response and simplicity in usage. The working characteristics of biosensors often depend on efficacy of a biological stuff immobilization on the surface of transducer. In this context there is a need for development of immobilization methods capable to provide for execution of the following demands: 1) compatibility of this process with technology of building transducer; 2) simplicity in fulfillment, cheapness and expressness at manufacture of biomembranes; 3) ability to provide the maximal safety of a biological stuff activity and its high adhesion to a surface of transducer; 4) reproducibility at serial application and capacity of standardizing. Usage of photocrosslinked and photopolymerized compound at the immobilization of a biological stuff allows to provide execution of the listed above demands. The present review is devoted to features of application of the given class of compound at building of biosensors.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.     

Optical probes and transducers

Biosensors are by definition a combination of a biological receptor compound and a physical or physicochemical transducer. Therefore, the transducing structure is a critical part of every biosensor. In the development of new and improved biosensing layers the importance of the transducing structure is not restricted to the substrate to which biological structures have to be coupled. A field of even greater importance is the use of transducers as probes providing information on the structure and function of biosensing layers, and their relation to a transducer surface.

The aim of this paper is to give an overview on optical transducer principles and optical (surface) analytical techniques relevant as part of biosensing structures as well as probes in the development and optimisation of biosensing layers. Categories discussed are basic optical effects, materials involved, surface chemistry, the principal and technological limits of spatial resolution, and sensitivity. The intimate relation between the spatial resolution of a probe, the resulting size of interaction areas, and the feasibility of array structures is pointed out.

Two interferometric methods are presented in principle, and their application to biosensing and some results are discussed in detail. The necessity to characterise receptor layers to get detailed information about the interaction process is pointed out. The close relationship between optimal characterisation of layers by selection of adequate probe technologies and improvement of probe performance, and the development of new biosensing layers is discussed. Finally an outlook is given for future aspects of improved spatial resolution and multianalyte detection.     

Biosensor-based label-free assays of amyloid growth

Uncontrolled fibrous protein aggregation is implicated in a range of aberrant biological phenomena. Much effort has consequently been directed towards establishing quantitative in vitro assays of this process with the aim of probing amyloid growth in molecular detail as well as elucidating the effect of additional species on this reaction. In this paper, we discuss some recent approaches based on label-free technologies focussed on achieving these objectives. Several biosensor techniques have been developed to monitor biomolecular assembly without the requirement for fluorophore marker molecules; in particular quartz crystal microbalance and surface plasmon resonance measurements provide advantageous alternatives to traditional spectroscopic methods and are currently receiving increasing attention in the context of amyloid growth assays.     

A potentiometric protein sensor built with surface molecular imprinting method

Surface molecular imprinting, as compared to molecular imprinted bulk polymers, has the advantages of higher re-occupation percentage of the reception sites, fast response, integration of sensing element and transducer, etc. In this study, a potentiometric protein sensor was developed based on the surface molecular imprinting technique. Using the self-assembled monolayers of alkanethiol with hydroxyl terminal groups as the matrix material, and target protein molecules as the template, the sensing layer was created on the surface of the gold-coated silicon chip-an electrochemical transducer. Potentiometric measurement demonstrated that the sensor could selectively detect myoglobin or hemoglobin molecules, either with or without the presence of other protein molecules in the same solution.     

A streptavidin surface on planar glass substrates for the detection of biomolecular interaction

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved.     

DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging

Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features.     

Modulators of neuronal migration and neurite growth.

A multitude of molecules have been identified over the past few years that promote neurite outgrowth in vitro. The concept that these molecules work mainly by providing an adhesive surface for neuronal growth cones has been challenged by evidence from recent experiments. Some of the substrate molecules have diverse actions on cell migration and neurite growth. In addition, there is now evidence that there are molecules that specifically inhibit growth cone locomotion. This has given rise to the hypothesis that growth cones integrate a variety of growth-promoting and inhibitory signals and translate them into directed locomotion.     

Biosensors: current applications and future potential

Biological molecules such as enzymes and antibodies display a unique capacity to recognize and respond to other molecules in a way which can be exploited in the development of analytical devices. In a biosensor, the biological recognition system creates a physiochemical change proximal to a suitable transducer and thereby converts the concentration of the analyte into a quantifiable electrical signal. The design and construction of these devices requires an imaginative combination of biological, chemical, physical and engineering disciplines. Biosensors will find application in a variety of analytical fields.     

Role of tumor-associated gangliosides in cancer progression

Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted.     

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.     

Major histocompatibility complex (MHC) class II: are lipid rafts the missing link?

Aside from their crucial roles in the presentation of nominal antigen to CD4+ T cells and susceptibility to autoimmune diseases, substantial evidences suggest that MHC class II molecules act as signal transducer receptors as well. The signals transmitted affect diverse biological functions. Paradoxically, the cytoplasmic and transmembrane domains of these molecules are devoid of classic signaling motifs. The recent discovery of the presence of membrane microdomains, also called lipid rafts, that are enriched in kinases and adaptor molecules, may contribute to the elucidation of the mechanisms by which MHC class II molecules transmit their signals.     

A Markovian engine for a biological energy transducer: the catalytic wheel

The molecular machines in biological cells are made of proteins, DNAs and other classes of molecules. The structures of these molecules are characteristically "soft", highly flexible, and yet their interactions with other molecules or ions are specific and selective. This chapter discusses a prevalent form, the catalytic wheel, or the energy transducer of cells, examines its mechanism of action, and extracts from it a set of simple but general rules for understanding the energetics of the biomolecular devices. These rules should also benefit design of manmade nanometer scale machines such as rotary motors or track-guided linear transporters. We will focus on an electric work that, by matching system dynamics and then enhancing the conformational fluctuation of one or several driver proteins, converts stochastic input of energy into rotation or locomotion of a receptor protein. The spatial (or barrier) and temporal symmetry breakings required for selected driver/receptor combinations are examined. This electric ratchet consists of a core engine that follows the Markovian dynamic, alleviates difficulties encountered in rigid mechanical model, and tailors to the soft-matter characteristics of the biomolecules.     

New tools for malaria therapy

In the absence of vaccines, new chemotherapies are needed urgently to help in the prevention and control of malaria. A number of strategies are being followed world-wide in attempts to discover and develop them: (a) resurrecting 'forgotten' molecules; (b) developing new formulations of existing products; (c) finding ways to bypass known toxicological limitations of existing products; (d) looking for combinations of existing products; (e) discovering molecules which reverse the resistance phenotype; (f) identifying 'old' chemical entities (OCEs) for new, antimalarial, indications; (g) discovering new chemical entities (NCEs) directed towards already exploited biological targets; (h) discovering NCEs directed to novel biological targets. Examples of such strategies are given below, together with an indication of their advantages and limitations.     

Reduced graphene oxide directed self-assembly of phospholipid monolayers in liquid and gel phases

The response of cell membranes to the local physical environment significantly determines many biological processes and the practical applications of biomaterials. A better understanding of the dynamic assembly and environmental response of lipid membranes can help understand these processes and design novel nanomaterials for biomedical applications. The present work demonstrates the directed assembly of lipid monolayers, in both liquid and gel phases, on the surface of a monolayered reduced graphene oxide (rGO). The results from atomic force microscopy indicate that the hydrophobic aromatic plane and the defect holes due to reduction of GO sheets, along with the phase state and planar surface pressure of lipids, corporately determine the morphology and lateral structure of the assembled lipid monolayers. The DOPC molecules, in liquid phase, probably spread over the rGO surface with their tails associating closely with the hydrophobic aromatic plane, and accumulate to form circles of high area surrounding the defect holes on rGO sheets. However, the DPPC molecules, in gel phase, prefer to form a layer of continuous membrane covering the whole rGO sheet including defect holes. The strong association between rGO sheets and lipid tails further influences the melting behavior of lipids. This work reveals a dramatic effect of the local structure and surface property of rGO sheets on the substrate-directed assembly and subsequent phase behavior of the supported lipid membranes.