New multi-step kinetics using common affinity biosensors saves time and sample at full access to kinetics and concentration

Today, affinity-based biosensorics is a standard technology in quantitative biomolecular interaction analysis, but suffers from low sample throughput and sometimes from inaccessible kinetics. A new methodology for such biosensors is introduced here that cuts down measurement time dramatically and increases confidentiality of results. In contrast to traditional applications, the ligand immobilized on the sensor chip is exposed to the binding analyte at a rapid stepwise change of the analyte concentration without the need for regenerations between analyte additions. In the application presented here, each addition of the analyte is succeeded by a buffer flow, yielding alternating association and dissociation phases in a "zigzag" style. This binding curve pattern is analyzed by means of novel fitting algorithms, which render detailed kinetics rate constants at a high level of self-consistency, and hence, validity due to multiple cross-checks. In comparison with traditional sequential kinetics analysis, this new multi-step kinetics approach returns practically identical (or improved) kinetics constants--at valuable savings in time/material since regeneration steps, ligand re-captures, or titration equilibrations are unnecessary.     

Reconstruction of the antibody affinity distribution from experimental binding data by a minimum cross-entropy procedure.

A new solution is presented for the reconstruction of the distribution of association constants of antigen-antibody binding from a finite number of noisy experimental binding data. This ill-posed problem is solved by utilizing an information-theoretic method based on the principle of minimum cross-entropy (MCE) to select, as the solution, that unique antibody binding distribution which minimizes the cross entropy relative to some prior distribution subject to the constraints imposed by the given measurements. The prior distribution is selected to properly encode all the a priori information on the affinity distribution before the measurement of the experimental binding data. The utility of the method is demonstrated by application to synthetic binding data.     

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

Background  

Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.     

A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using "differential dissociation" techniques.

The steroid hormone-receptor complex concentrations measured by “differential dissociation” techniques have to be corrected to obtain the true concentrations of receptor binding sites (B_s). For the calculation of B_s, the parameters kn (product of the equilibrium association constant and the concentration of binding sites of the “nonspecific” component) and f (fraction of the nonspecific binding measured in the experimental estimates of bound ligand by a given technique), previously proposed by Blondeau and Robel (J. P. Blondeau and P. Robel, 1975, Eur. J. Biochem.55, 375–384) are important. A new parameter of interest, ? [$\text{?}\text{=}\text{knf}\text{(kn + 1)}$], is discussed. The measurement of this parameter ? for three “differential dissociation” techniques allows the comparison of their efficiency and their reliability under various conditions for hormone receptor measurement in cytosol. Charcoal and hydroxylapatite methods are more efficient than the Sephadex G-25 filtration method. It is demonstrated that the “isotopic dilution” correction generally used for the estimation of the background of a given technique may be incorrect whatever the method of correction. A new method, the “double concentration measurement,” is developed. This method is simple, rapid, and precise. It requires two receptor binding measurements at two different saturating concentrations of ligand. This method allows the measurement of the estradiol receptor binding activity from calf uterine cytosol, with an error of less than 5% in samples containing the receptor either free or previously complexed with radioactive hormone, even in the presence of very high concentrations (≤0.5 μm) of radioactive steroid.     

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 μM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay.

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

Application of the miniature ultracentrifuge in receptor-binding assays.

A new binding assay for membrane receptor systems has been developed employing an air-driven ultracentrifuge (Beckman Airfuge). The main advantages of this method for measurement of radioligand binding in aqueous medium are (i) the rapidity (30 s) in separating the bound from the unbound fraction, (ii) the small volume (100 μl) of assay medium which permits a relatively small excess of ligand over receptor to be employed, and (iii) the simplicity of manipulations which allows a high degree of replication. The variation in a triplicate set of assays is usually less than 0.5%. By virtue of maintaining equilibrium throughout the assay the present method is especially useful for ligands exhibiting rapid reversibility in binding. Binding of [³H]ouabain to several membrane (Na⁺, K⁺)-ATPases and binding of [³H]etorphine to the oplate receptor from brain membranes are discussed here. Also the inhibition of [³H]ouabain binding by Tris is discussed.     

How is Pheromone Specificity Encoded in Proteins?

Pheromone specificity in the Lepidoptera is encoded in proteincomponents of the antennal sensillum lymph and dendritic membrane.In this paper, we highlight recent work on the molecular determinantsof pheromone binding affinity of pheromone binding proteins(PBPS) of three genera. First, we describe new cDNA sequencesfor Lymantria dispar (Lymantriidae) and Agrotis segetum (Noctuidae).These data enrich the conclusions derived from our functionalstudies. Secondly, we indicate how preparation of multimilligramquantities of the recombinant PBP ‘Apol-3’ (originallyfrom Antheraea polyphemus) has provided a platform (i) to determinethe ligand binding sites using photoaffinity labeling, (ii)to conduct structural analysis by CD and NMR, and (iii) to measurebinding affinities using a new binding assay. Thirdly, we describethe use of expression-cassette PCR technology to prepare tworelated PBPS from Antheraea perneyi to test binding affinitiesof naturally-occurring homologous PBPs. Our results supporta model in which ligand specificity for chain length, doublebond position, and terminal functionality is partially encodedin the PBPS. We propose that the final decoding is accomplishedwhen the PBP-pheromone complex activates a G-protein coupledseven-transmembrane domain receptor that contains recognitionsites for both the presented pheromone and the presenting PBP.Chem. Senses 20: 461–469, 1995.     

Calculation of z-coordinates and orientational restraints using a metal binding tag

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.     

A Novel Strategy for Analyzing RNA-Protein Interactions by Surface Plasmon Resonance Biosensor

Surface plasmon resonance (SPR) biosensor is a promising technology for its various advantages including the real-time measurement of biomolecular interactions without labeling. A method of hybridizing RNAs on the surface of the streptavidin-coated (SA) sensor chip to study RNA-protein interactions was described in this paper. In our study, it has been shown that the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has a high binding affinity for the leader sequence of SARS-CoV genome. Effect of temperature on the RNA-DNA hybridization was also examined. This method can provide the affinity of interactions with high sensitivity. Therefore, it will be useful in screening binding candidates for a given RNA target motif with one chip.     

A rapid and high‐throughput method for the determination of serum uric acid based on microarray technology and nanomaterial

Serum uric acid (SUA) is a new therapeutic target for non‐alcoholic fatty liver disease (NAFLD). In this study, we introduced a chemiluminescence (CL) method combined with microarray technology and a simple fabrication procedure to obtain a highly sensitive SUA probe based on a mesoporous metal oxide nanomaterial. The high‐throughput method was based on the generation of H₂O₂ from SUA by immobilized uricase and its measurement by a CL reaction catalyzed by mesoporous metal oxide nanomaterials. The CL probe was designed for SUA The linear range of the uric acid concentration was 0.6–9 μM and the detection limit was 0.1 μM. In comparison with the other SUA detection techniques, this method has the advantages of a low detection limit, high sensitivity and simplicity. A new sensitive high‐throughput approach was obtained for the determination of SUA.     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Antibody fragments from a ''single pot'' phage display library as immunochemical reagents.

The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to make immunochemical reagents to a range of antigens by selection from a repertoire of > 10(8) clones made in vitro from human V gene segments. From the same 'single pot' repertoire, phage were isolated with binding activities to each of 18 antigens, including the intracellular proteins p53, elongation factor EF-1 alpha, immunoglobulin binding protein, rhombotin-2 oncogene protein and sex determining region Y protein. Both phage and scFv fragments secreted from infected bacteria were used as monoclonal and polyclonal reagents in Western blots. Furthermore the monoclonal reagents were used for epitope mapping (a new epitope of p53 was identified) and for staining of cells. This shows that antibody reagents for research can be readily derived from 'single pot' phage display libraries.     

Exposure assessment of mobile phone base station radiation in an outdoor environment using sequential surrogate modeling

Human exposure to background radiofrequency electromagnetic fields (RF‐EMF) has been increasing with the introduction of new technologies. There is a definite need for the quantification of RF‐EMF exposure but a robust exposure assessment is not yet possible, mainly due to the lack of a fast and efficient measurement procedure. In this article, a new procedure is proposed for accurately mapping the exposure to base station radiation in an outdoor environment based on surrogate modeling and sequential design, an entirely new approach in the domain of dosimetry for human RF exposure. We tested our procedure in an urban area of about 0.04 km² for Global System for Mobile Communications (GSM) technology at 900 MHz (GSM900) using a personal exposimeter. Fifty measurement locations were sufficient to obtain a coarse street exposure map, locating regions of high and low exposure; 70 measurement locations were sufficient to characterize the electric field distribution in the area and build an accurate predictive interpolation model. Hence, accurate GSM900 downlink outdoor exposure maps (for use in, e.g., governmental risk communication and epidemiological studies) are developed by combining the proven efficiency of sequential design with the speed of exposimeter measurements and their ease of handling. Bioelectromagnetics 34:300–311, 2013. © 2012 Wiley Periodicals, Inc.     

Inertial sensor based method for identifying spherical joint center of rotation

The miniaturized wireless inertial measurement unit (IMU) technology and algorithms presented herein promote rapid and accurate predictions of the center-of-rotation (CoR) for ball/spherical joints. The algorithm improves upon existing IMU-based methods by directly utilizing the measured acceleration and angular velocity provided by the IMU to deduce the CoR in lieu of relying on error-prone velocity and position estimates. Results demonstrate that this new method resolves the position of the CoR to within a 3 mm sphere of the true CoR determined by a precision coordinate measuring machine. Such accuracy may render this method attractive for broad use in field, laboratory and clinical settings requiring non-invasive and rapid estimates of joint CoR.     

Profiling of cytokine expression by biotin-labeled-based protein arrays

Global analysis of protein expression holds great promise in basic research and patient care. Previously we demonstrated that multiple cytokines could be detected simultaneously using an enzyme-linked immunosorbent assay protein array system with high sensitivity and specificity. In this paper, we described a biotin-labeled-based protein array system to detect multiple cytokines simultaneously from biological samples. In this new approach, proteins from a variety of biological sources are labeled with biotin. The biotin-labeled proteins are then incubated with antibody chips. Targeted proteins are captured by the array antibodies spotted on the antibody chips. The presence of targeted proteins is detected using Cy3- or Cy5-conjugated streptavidin and signals are imaged by laser scanner. The system also can be easily adapted to a two-color binding assay, allowing measurement of the levels of proteins in a test sample with respect to a reference sample at the same chip. To demonstrate its potential applications, we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in estrogen receptor (ER)+ and ER- cells. These results suggest that biotin-labeled-based antibody chip technology can provide a practical and powerful means of profiling hundreds or thousands of proteins for research and clinical purposes.     

Binding analyses between Human PPARgamma-LBD and ligands.

The binding characteristics of a series of PPARgamma ligands (GW9662, GI 262570, cis-parinaric acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), LY171883, indomethacin, linoleic acid, palmitic acid and troglitazone) to human PPARgamma ligand binding domain have been investigated for the first time by using surface plasmon resonance biosensor technology, CD spectroscopy and molecular docking simulation. The surface plasmon resonance biosensor determined equilibrium dissociation constants (KD values) are in agreement with the results reported in the literature measured by other methods, indicating that the surface plasmon resonance biosensor can assume a direct assay method in screening new PPARgamma agonists or antagonists. Conformational changes of PPARgamma caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (T(m)), was enhanced by the binding of the ligands. The increment of the transition temperature (DeltaT(m)) of PPARgamma owing to ligand binding correlated well with the binding affinity. This finding implies that CD could possibly be a complementary technology with which to determine the binding affinities of ligands to PPARgamma. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARgamma at the atomic level, which gave a good explanation of the structure-binding affinity relationship for the ligands interacting with PPARgamma. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARgamma ligands, although the docking program cannot accurately predict the absolute ligand-PPARgamma binding affinity.