Modelling of multi-component immunoassay kinetics — A new node-based method for simulation of complex assays

The behaviour of binding reactions in immunoassays can be predicted and studied by modelling methods. Simple antibody-analyte binding reaction kinetics can be simulated by e.g. a mechanistic assay model based on differential equations. However, the mathematical modelling becomes more complicated if multivalent-structured components are involved and the number of binding complexes increases.In this paper, a new node-based method to model complex binding reactions is introduced. The principle of this method is to construct a network of the initial components, reaction intermediates and end-products by forming a network of nodes. This network is then solved, node by node, breaking the initial problem into smaller partial problems, still obeying the laws of chemical reaction kinetics and without ignoring any parts of the problem.This method provides an easy and quick way to study complex binding reactions since simulation networks are simple to construct directly from the reaction scheme. This presented new “NODE”-method is compared with the well known mechanistic assay model.     

Calibration of measured center of pressure of a new stairway design for kinetic analysis of stair climbing

A stairway that allows the collection of kinetic data is essential for biomechanical studies on stair climbing. There is a need to validate the measured center of pressure (COP) on the surface of a stair in order to verify the accuracy of the calculation of joint kinetics. The purpose of this study was to validate a new stairway design for kinetic analysis of stair climbing through a calibration and error analysis of the COP obtained from this system. The new stairway design allows the collection of kinetic data for multiple steps without any constraint to foot placement. Known vertical forces were applied to known locations on the surface of each stair and each force plate. Multiple regression analyses were conduced to determine the distribution pattern of the error in the measured COP. It was found that the error in the COP was a function of location on the stair or force plate. The magnitude of the vertical force had no significant effect on the error in the measured COP. The distribution pattern of the error in the measured COP on the force plates used in this study matched the results in the literature. A healthy female subject was used as the subject in a stair climbing test. The error in the measured COP had a significant effect on the calculated joint resultant moments, especially the abduction-adduction and internal-external rotation moments. The correction of these errors should make the kinetic calculation in stair climbing more accurate.     

Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism

The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.     

A kinetic model for an autopoietic synthesis of micelles

An improved solvable kinetic model for the autocatalytic production of micelles via the basic hydrolysis of ethyl caprylic ester is presented. A good agreement with the experimental results is obtained, in particular a second observable of the system, the micellar concentration is correctly forseen by the improved model.     

The interpretation of multiple-step transient-state kinetic isotope effects

In contrast to steady-state kinetic isotope effects (KIEs), transient-state tKIEs are both time and signal dependent and therefore require a very different form of theory for their interpretation. We have previously provided such a theory for the case of single-step isotopic substitutions. No such properly derived theory applicable to the analysis of multiple-step isotopic substitutions required by transient-state solvent isotope effect studies has been available up to this time. Here, we set forth a more general form of that theory which is applicable to multiple-step substituted cases. We prove three theorems: 1. the observed transient-state KIE for any given reactive component in the reaction sequence evaluated at zero time (tKIE(0)) is in fact the arithmetic product of the intrinsic KIEs of all the steps that precede the formation of that component. 2. The observed tKIE(0) is completely independent of the intrinsic KIEs of any reverse step in the reaction. 3. The intrinsic KIE of any step may be obtained by dividing the value of the tKIE(0) for that step by the value of the tKIE(0) of the immediately preceding step in the reaction sequence.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

New developments in CLAMP: Calibration using global gridded meteorological data

Climate Leaf Analysis Multivariate Program (CLAMP) is a versatile technique for obtaining quantitative estimates for multiple terrestrial palaeoclimate variables from woody dicot leaf assemblages. To date it has been most widely applied to the Late Cretaceous and Tertiary of the mid- to high latitudes because of concerns over the relative dearth of calibration sites in modern low-latitude warm climates, and the loss of information associated with the lack of marginal teeth on leaves in paratropical to tropical vegetation. This limits CLAMP's ability to quantify reliably climates at low latitudes in greenhouse worlds of the past.One of the reasons for the lack of CLAMP calibration samples from warm environments is the paucity of climate stations close to potential calibration vegetation sites at low latitudes. Agriculture and urban development have destroyed most lowland sites and natural vegetation is now largely confined to mountainous areas where climate stations are few and climatic spatial variation is high due to topographic complexity. To attempt to overcome this we have utilised a 0.5° × 0.5° grid of global interpolated climate data based on the data set of New et al. (1999) supplemented by the ERA40 re-analysis data for atmospheric temperature at upper levels. For each location, the 3-D climatology of temperature from the ECMWF re-analysis project was used to calculate the mean lower tropospheric lapse rate for each month of the year. The gridded data were then corrected to the altitude of the plant site using the monthly lapse rates. Corrections for humidity were also made. From this the commonly returned CLAMP climate variables were calculated. A bilinear interpolation scheme was then used to calculate the climate parameters at the exact lat/long of the site.When CLAMP analyses using the PHYSG3BR physiognomic data calibrated with the climate station based MET3BR were compared to analyses using the gridded data at the same locations (GRIDMET3BR), the results were indistinguishable in that they fell within the range of statistical uncertainty determined for each analysis. This opens the way to including natural vegetation anywhere in the world irrespective of the proximity of a meteorological station.     

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.     

Analysis of kinetic,stoichiometry and regulation of glucose and glutamine metabolism in hybridoma batch cultures using logistic equations

Batch cultures were carried out to study the kinetic, stoichiometry, and regulation of glucose and glutamine metabolism of a murine hybridoma line. Asymmetric logistic equations (ALEs) were used to fit total and viable cell density, and nutrient and metabolite/product concentrations. Since these equations were analytically differentiable, specific rates and yield coefficients were readily calculated. Asymmetric logistic equations described satisfactorily uncontrolled batch cultures, including death phase. Specific growth rate showed a Monod-type dependence on initial glucose and glutamine concentrations. Yield coefficients of cell and lactate from glucose, and cell and ammonium from glutamine were all found to change dramatically at low residual glucose and glutamine concentrations. Under stoichiometric glucose limitation, the glucose-to-cell yield increased and glucose-to-lactate yield decreased, indicating a metabolic shift. Under stoichiometric glutamine limitation the glutamine-to-cell and glutamine-to-ammonium yields increased, but also glucose-to-cell yield increased and the glucose-to-lactate yield decreased. Monoclonal antibody production was mainly non-growth associated, independently of glucose and glutamine levels.     

Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)-dependent dehydrogenases using the kinetic locking-on strategy.

In preparation for studies aimed at establishing the relationship between immobilized NAD(+) concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD(+)-dependent dehydrogenases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD(+) concentration. Affinity chromatographic studies using S(6)-linked NAD(+) derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD(+) concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.     

A mathematical model of lateral flow bioreactions applied to sandwich assays

Lateral flow (LF) biodetectors facilitate low-cost, rapid identification of various analytes at the point of care. The LF cell consists of a porous membrane containing immobilized ligands at various locations. Through the action of capillary forces, samples and reporter particles are transported to the ligand sites. The LF membrane is then scanned or probed, and the concentration of reporter particles is measured. A mathematical model for sandwich assays is constructed and used to study the performance of the LF device under various operating conditions. The model provides insights into certain experimental observations including the reduction in the level of the detected signal at high target analyte concentrations. Furthermore, the model can be used to test rapidly and inexpensively various operating conditions, assist in the device's design, and optimize the performance of the LF device.     

Kinematic and kinetic comparison of baseball pitching among various levels of development

Proper biomechanics help baseball pitchers minimize their risk of injury and maximize performance. However previous studies involved adult pitchers only. In this study, 23 youth, 33 high school, 115 college, and 60 professional baseball pitchers were analyzed. Sixteen kinematic (11 position and five velocity), eight kinetic, and six temporal parameters were calculated and compared among the four levels of competition. Only one of the 11 kinematic position parameters showed significant differences among the four levels, while all five velocity parameters showed significant differences. All eight kinetic parameters increased significantly with competition level. None of the six temporal parameters showed significant differences. Since 16 of the 17 position and temporal parameters showed no significant differences, this study supports the philosophy that a child should be taught ‘proper’ pitching mechanics for use throughout a career. Kinetic differences observed suggest greater injury risk at higher competition levels. Since adult pitchers did not demonstrate different position or temporal patterns than younger pitchers, increases in joint forces and torques were most likely due to increased strength and muscle mass in the higher level athlete. The greater shoulder and elbow angular velocities produced by high-level pitchers were most likely due to the greater torques they generated during the arm cocking and acceleration phases. The combination of more arm angular velocity and a longer arm resulted in greater linear ball velocity for the higher level pitcher. Thus, it appears that the natural progression for successful pitching is to learn proper mechanics as early as possible, and build strength as the body matures.     

Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.     

TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V_max of 41.4 ± 3.3 nM/min and K_M of 1.60 ± 0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.     

The choice of reference cell in the analysis of kinetic data using BIAcore.

An important step in the analysis of sensorgram data for BIAcore experiments is the subtraction of reference cell data to remove the effects of the bulk shift on the sensorgram of interest. It is shown that this step can introduce errors in the measured kinetic constants. This phenomenon is investigated both theoretically and with experimental data.     

High-efficiency bioaffinity separation of cells and proteins using novel thermoresponsive biotinylated magnetic nanoparticles

Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max, avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4 antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max with a UCST in bioaffinity separation of cells and biomolecules.     

A kinetic study on the chemical cleavage of nucleoside diphosphate sugars

Nucleoside diphosphate sugars serve in essential roles in metabolic processes. They have, therefore, been used in mechanistic studies on glycosylation reactions, and their analogues have been synthesised as enzyme and receptor inhibitors. Despite extensive biochemical research, little is known about their chemical reactions. In the present work the chemical cleavage of two different types of nucleoside diphosphate sugars has been studied. UDP-Glc is phosphorylated at the anomeric carbon, whereas in ADP-Rib C-1 is unsubstituted, allowing hence the equilibrium between cyclic hemiacetal and acyclic carbonyl forms. Due to the structural difference, these substrates react via different pathways under slightly alkaline conditions: while UDP-Glc reacts exclusively by a nucleophilic attack of a glucose hydroxyl group on the diphosphate moiety, ADP-Rib undergoes a complex reaction sequence that involves isomerisation processes of the acyclic ribose sugar and results in a release of ADP.     

Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers

A weighted nonlinear least-squares curve-fitting program, implemented in compiled BASIC for the IBM-PC is described to estimate the parameters of enzyme kinetics obeying Michaelis-Menten kinetics and seven inhibition models. The effects of the inhibitor on the maximal velocity (Vm) and the Michaelis-Menten constant (Km) are used to select automatically the most plausible model of inhibition and to calculate initial estimates of parameters. The program is used to demonstrate that the inhibition of carbamyl-phenylalanine hydrolase by the product phenylalanine is consistent with the pure mixed noncompetitive model.     

Denoising array-based comparative genomic hybridization data using wavelets

Array-based comparative genomic hybridization (array-CGH) provides a high-throughput, high-resolution method to measure relative changes in DNA copy number simultaneously at thousands of genomic loci. Typically, these measurements are reported and displayed linearly on chromosome maps, and gains and losses are detected as deviations from normal diploid cells. We propose that one may consider denoising the data to uncover the true copy number changes before drawing inferences on the patterns of aberrations in the samples. Nonparametric techniques are particularly suitable for data denoising as they do not impose a parametric model in finding structures in the data. In this paper, we employ wavelets to denoise the data as wavelets have sound theoretical properties and a fast computational algorithm, and are particularly well suited for handling the abrupt changes seen in array-CGH data. A simulation study shows that denoising data prior to testing can achieve greater power in detecting the aberrant spot than using the raw data without denoising. Finally, we illustrate the method on two array-CGH data sets.