Specific Antigen-antibody Binding Research Articles

Development of a Surface Plasmon Resonance Biosensor for Real-Time Detection of Osteogenic Differentiation in Live Mesenchymal Stem Cells

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

Fully Distributed Fiber-Optic Biological Sensing

Fully distributed fiber-optic biological sensing is demonstrated based on a traveling long-period grating (LPG) in a single-mode fiber coated with a functional film. The temporal LPG is generated by an acoustic pulse propagating along the fiber. Immunoglobulin G (IgG) is immobilized onto the surface of the fiber via ionic self-assembly. We show that specific antigen-antibody binding can introduce a measurable shift in the transmission optical spectrum of the traveling LPG when it passes through the 10-cm pretreated fiber segment. It is also shown that nonspecific binding to other unintended IgG can be avoided by the application of a standard binding block in the sensor fabrication. The experimental result proves that the traveling LPG-based fiber sensing method offers an attractive means for fully distributed bio sensing. This method may also permit measurement of other quantities that can shift the transmission spectrum of the traveling LPG by changing the properties of the fiber itself or by the assistance of a functional coating on the surface of the fiber.

Nanopyramid surface plasmon resonance sensors

We report the achievement of sensitive chemical and biological sensing using periodic gold nanopyramids with nanoscale sharp tips created by a simple and scalable colloidal templating approach. The sharp tips and the long-range periodic structure of the nanopyramid arrays enable the excitement of both localized and propagating surface plasmons. The optical reflection and the detection sensitivity of the templated nanopyramid surface plasmon resonance sensors agree reasonably well with the theoretical predictions using a finite-difference time-domain model. We have also demonstrated that specific antigen-antibody binding can be detected by using nanopyramid arrays in a real-time and label-free manner.

Avoiding Nonspecific Interactions in Studies of the Plasma Proteome: Practical Solutions to Prevention of Nonspecific Interactions for Label-Free Detection of Low-Abundance Plasma Proteins

The molecular constitution of blood can be highly representative of the physiological state of an individual and offers an ideal target for studies of biomarkers. High-abundance plasma proteins, particularly albumin, dominate the plasma proteome, but it is the low-abundance proteins (such as cytokines) that are commonly associated with many pathophysiological states. Several detection strategies, and particularly those that involve label-free detection, are available for low-abundance protein detection in plasma, but all can be severely compromised by the high-abundance of serum albumin. In the present study, we examine the effect of albumin interference on accurate label-free detection by protein microarrays. Albumin was found to disrupt specific antigen-antibody binding interactions of low-abundance proteins. In clinical analysis, where it is imperative to preserve the integrity of samples, depletion of albumin may further undermine quantitative measurements. We have optimized procedures that permit accurate analysis to be undertaken without the need for prior treatment of samples. The emphasis is placed on disrupting nonspecific interactions including both electrostatic (i.e., Colulombic) and electrodynamic (hydrophobic and other nonpolar based) interactions. These protocols appear to be generic with potential applications in several areas of analytical biotechnology.

Fast and Sensitive Measurement of Specific Antigen-Antibody Binding Reactions With Magnetic Nanoparticles and HTS SQUID

We have developed an HTS SQUID system for both time and frequency domain measurements of magnetic signals from the Brownian relaxation of liquid-suspended magnetic nanoparticles (MNPs) coated with antibodies. A planar YBa <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> Cu <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">7-x</sub> dc SQUID gradiometer with a baseline of 3 mm allows stable operation of the measurement system in an unshielded environment. Agglomeration of MNPs induced by prostate specific antigen-antibody binding complexes is characterized with our system and benchmarked with frequency domain measurements performed by Imego AB using an induction coil magnetometer. We estimate an upper bound for our immunoassay analyte sensitivity of 1.8 ng/radicHz.

Monoklonale Antikörper

The use of monoclonal antibodies for therapeutic purposes and diagnostic imaging has become an important pillar of internal medicine in recent years. Currently, the application of antibodies against cell surface receptors and cytokines makes specific therapies possible, in particular against chronic and malignant diseases. The production of antibody preparations increasingly involves gene and biotechnological processes while continuing to adhere to the basic principle of specific antigen-antibody binding. Despite the complex antigen structure of antibodies, severe treatment side effects remain the exception. This is all the more remarkable considering the increased use of human or humanized and functionally optimized antibodies. In the coming years, numerous new monoclonal antibodies will extend the therapeutic and diagnostic possibilities for diverse clinical applications.

Quantitative Label-free Biodetection of Acute Disease Related Proteins Based on Nanomechanical Dynamic Microcantilevers

We report the label-free biomolecules detection based on nanomechanical microcantilevers operated in dynamic mode for detection of two marker proteins (myoglobin and creatin kinase-MB (CK-MB)) of acute myocardical infarctions. When the specific binding between the antigen and its antibody occurred on the fuctionalized microcantilever surface, mechanical response (i.e. resonant frequency) of microcantilevers was changed in lower frequency range. We performed the label-free biomolecules detection of myoglobin and CK-MB antigen in the low concentration (clinical threshold concentration range) as much as 1 ng/ml from measuring the dynamic response change of microcantilevers caused by the intermolecular force. Moreover, we estimate the surface stress on the dynamic microcantilevers generated by specific antibody-antigen binding. It is suggested that our dynamic microcantilevers may enable one to use the sensitive label-free biomolecules detection for application to the disease diagnosis system based on mechanical immuno-sensor.

Fibre-optic interferometric immuno-sensor using long period grating

A species-specific biosensor is presented based on the combined use of a reflection mode long period fibre grating with nanoscale self-assembly. Self-assembled polyelectrolyte layers and the immobilisation of immunoglobulin G (IgG) were used for sensor fabrication. The sensor&apos;s fringe position shift induced by specific antigen-antibody binding has been observed, and non-specific binding was minimised with binding block. The results show the feasibility of the sensor for various bio/chemical applications.

A resistive-pulse sensor chip for multianalyte immunoassays

MultiAnalyte immunoassays are often required to diagnose a pathologic condition. Here, we show how resistive-pulse sensing and multiple artificial pores can be integrated together on a single chip to detect different antigens rapidly and simultaneously. We use multiple pores on a single chip to detect the size change of latex colloids upon specific antigen-antibody binding on the colloid surface. As a proof-of-principle, we demonstrate our ability to detect simultaneously human G-CSF and GM-CSF antigens on a single chip. Our novel technique is a scalable technology that can lead to the sensing of at least N2 antigens simultaneously with an N N array of pores on a single chip.

Influence of Surfactants and Antibody Immobilization Strategy on Reducing Nonspecific Protein Interactions for Molecular Recognition Force Microscopy

Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.

Electrostatics in protein binding and function.

Protein electrostatic properties stem from the proportion and distribution of polar and charged residues. Polar and charged residues regulate the electrostatic properties by forming short-range interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein. Electrostatics play a major role in defining the mechanisms of protein-protein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements. For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions; Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts; Increase in binding specificity and affinity involve optimization of electrostatics; High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens; Rigid parts of proteins have higher and stronger electrostatic interactions. In this review we address the significance of electrostatics in protein folding, binding and function. We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function. We also provide bona fide examples to illustrate this. Additionally, using continuum electrostatic and molecular dynamics approaches we show that the "hot-spot" inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues. These "hot-spot" molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear. This further corroborates the significance of charge-charge interactions in defining binding mechanisms. High affinity binding frequently involves "electrostatic steering". The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions. We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases. These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.

Evanescent wave long-period fiber bragg grating as an immobilized antibody biosensor.

An immunosensor using a long-period grating (LPG) was used for sensitive detection of antibody-antigen reactions. Goat anti-human IgG (antibody) was immobilized on the surface of the LPG, and detection of specific antibody-antigen binding was investigated. This sensor operates using total internal reflection where an evanescent field interacts with bound antibody immobilized over the grating region. The reaction between antibody and antigen altered the LPG transmission spectrum and was monitored in real time as a change in refractive index, thereby eliminating the need for labeling antigen molecules. Human IgG binding was observed to be concentration dependent over a range of 2-100 microg mL-1, and equilibrium bound antigen levels could be attained in approximately 5 min using an initial rate determination. Binding specificity was confirmed using human interleukin-2 and bovine serum albumin as controls, and nonspecific adsorption of proteins did not significantly interfere with detection of binding. Antigen detection in a heterogeneous protein mixture and in crude cell lysate from Escherichia coli was also confirmed. Moreover, regeneration of the LPG surface via diethylamine treatment resulted in approximately 80% removal of bound antigen. Subsequently, fibers reexposed to antigen retained greater than 85% of the initial signal after five consecutive regeneration cycles.

Enzyme-linked immunosorbent assay of peptides.

AbstractEnzyme-linked immunosorbent assay (ELISA) is a member of the solid-phase immunoassay family that detects specific antigen-antibody binding reactions (1,2). A great many variables of antigen or antibody presentation, treatment, and detection are considered when performing ELISA (3,4). This chapter will describe the simplest form, a direct assay, in which the peptide antigen is immobilized on a polystyrene carrier, and a specific primary antibody in solution (detector) is added that recognizes the plate-bound antigen. A secondary, horseradish peroxidase (HRP)-coupled polyclonal antibody (reporter) binds to the primary antibody. The enzymatic activation of hydrogen peroxide releases a chromophore from o-phenylenediamine that is detected and quantified.KeywordsPeptide AntigenSodium FluorideSpecific Primary AntibodyAntibody PreparationSecondary Antibody SolutionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

High-sensitivity biosensor based on LB technology and on nanogravimetry

Quartz-resonator nanobalances are utilized here as effective transducers capable of determining with high sensitivity the mass changes due to specific protein-protein, antigen-antibody and ligand-receptor binding or self-assembly of functional complexes. The resulting highly sensitive biosensor is based on two quartz resonators (one active and the second used as a reference) and on Langmuir-Blodgett (LB) protein monolayers. The electronics are composed of two separate blocks, one designed to acquire by a personal computer the data coming from the other card, a 24-bit digital counter directly connected to the two oscillators. In the case of immunosensor application, the active and reference oscillators are covered respectively by antibodies specific to a given antigen and by antibodies non-specific to the antigen, in order to disciminate the physical adsorption effects. Deposition of antibody monolayers is performed by the LB technique in a surface pressure range of 20–35 mN m −1 onto gluteraldehyde pre-treated quartz resonators. A thermal treatment of the antibody layer up to 150 °C results in the reorganization of the film, and significantly improves the sensitivity and the properties of the immunosensor.

Impedance based sensing of the specific binding reaction between Staphylococcus enterotoxin B and its antibody on an ultra-thin platinum film

Immunobiosensing techniques to measure specific antigen-antibody binding reactions are important in the development of biosensor applications in biotechnology, in vitro diagnosis, medicine and food technology. An immunobiosensor was constructed to measure the specific binding reaction between Staphylococcus enterotoxin B (SEB) and anti-SEB antibodies. The biosensor comprised an anti-SEB bioactive layer covalently immobilized on an ultra-thin platinum (Pt) film sputtered onto a 100 nm thick silicon dioxide layer on a silicon chip. The Pt film was discontinuous with a normal thickness of 25 Å. The impedance of the Pt film decreased during the binding of the anti-SEB to SEB in phosphate buffered saline (PBS) at room temperature. The impedance decreases were irreversible in PBS before saturation of the specific binding sites. When saturated, the impedance at 100 Hz was 14% of the value obtained for the fresh anti-SEB layer in PBS. The magnitude of the impedance (∥Z∥) decrease followed a simple relationship with SEB concentration in the range between 0.389 and 10.70 ng/ml SEB. The specificity of the biosensor was demonstrated by showing that no irreversible impedance decreases occurred when the sensor was exposed to 100 ng/ml κ-casein, α-casein, or α-lactalbumin, in PBS.