Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.     

Plasma carnitine and acetyl-carnitine levels at different times of the day

An interest in both biochemical and clinical carnitine investigation has recently developed. A more complete and extensive study is obtained if acetyl-carnitine as well as carnitine are investigated. This research, using an improved and simplified method for carnitine and acetyl-carnitine determination in the same sample (1 ml) without radioisotopic tracer use, investigates if there are the same differences in their plasma levels at different times of the day. The sample was eluted in a chromatographic column (55 X 15 mm) containing Sephadex G-25M with phosphate buffer (25 mmol/l, pH 7.4). The fraction containing acetyl and free carnitine was divided and employed separately for two assays. The carnitine assay uses an enzymatic reaction catalyzed by carnitine acetyl-transferase (CAT) and measurements are carried out spectrophotometrically. The calibration curve shows r = 0.987 and sensitivity at 5 mumol/l (reference plasma values: 38 +/- 3 mumol/l in 9 subjects). The acetyl-carnitine assay is carried out concentrating the sample by lyophilization and then measuring the enzymatic coupled reactions catalyzed by CAT, malate dehydrogenase and citrate synthase fluorimetrically. The calibration curve gives r = 0.991 and sensitivity at 1.4 mumol/l (reference plasma values: 2.8 +/- 0.3 mumol/l in 9 subjects). Both assay methods are measured at the end point. The carnitine and acetyl-carnitine measured in the plasma of 6 normal subjects at different times of the day vary respectively from 28 to 37 mumol/l and from 1.1 to 5.2 mumol/l in agreement with plasma free fatty acid (FFA) variation from 230 to 779 microEq/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical assessments of errors in rapid immunoassays-how critical is the exact timing and reagent concentrations?

The reliability of rapid immunoassay is a concern due to an incomplete incubation to a non-equilibrium state and is susceptible to different error factors causing variance. The most critical point in the process should be found in order to improve the accuracy, and reproducibility of immunoassays, and enhance the system robustness. In this paper, the behavior of rapid assays is predicted by simulations using mechanistic assay model, based on antibody-analyte binding reaction kinetics. This antibody-analyte binding reaction kinetics model was constructed for a generic three-component (immunometric) assay and the parameters were chosen to be those of a known surface binding assay. The effects of the exact incubation timing and the initial reagent concentrations were studied focusing on the early phase of incubation, the non-equilibrium state. The magnitudes of errors in the input parameters were estimated using knowledge from practical immunoassays. According to simulations, inaccurate incubation timing adds error in the results at very short incubation times, especially in low analyte concentrations. The inaccurate reagent concentrations increase variance at short incubation times, as well. The error decreases rapidly after the first few minutes of incubation.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

PSysCal: a parallel tool for calibration of ecosystem models

The methods used for ecosystem modelling are generally based on differential equations. Nowadays, new computational models based on concurrent processing of multiple agents (multi-agents) or the simulation of biological processes with the Population Dynamic P-System models (PDPs) are gaining importance. These models have significant advantages over traditional models, such as high computational efficiency, modularity and its ability to model the interaction between different biological processes which operate concurrently. By this, they are becoming useful for simulating complex dynamic ecosystems, untreatable with classical techniques. On the other hand, the main counterpart of P-System models is the need for calibration. The model parameters represent the field measurements taken by experts. However, the exact values of some of these parameters are unknown and experts define a numerical interval of possible values. Therefore, it is necessary to perform a calibration process to fit the best value of each interval. When the number of unknown parameters increases, the calibration process becomes computationally complex and storage requirements increase significantly. In this paper, we present a parallel tool (PSysCal) for calibrating next generation PDP models. The results shown that the calibration time is reduced exponentially with the amount of computational resources. However, the complexity of the calibration process and a limitation in the number of available computational resources make the calibration process intractable for large models. To solve this, we propose a heuristic technique (PSysCal+H). The results show that this technique significantly reduces the computational cost, it being practical for solving large model instances even with limited computational resources.     

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.     

Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z' factor of 0.78.     

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.     

Quantitative label-free screening for antibodies using scattering biophotonic microarray imaging

A biophotonic array based on gold nanoparticles functionalized with antigen proteins has been used to determine the concentrations of the respective antibodies in solution. Four proteins—fibrinogen, bovine serum albumin, transferrin, and C-reactive protein—were used to construct a test array with the assay repeated a number of times. The antibody-antigen association and dissociation rate constants were determined for the antibody assays from a series of calibration experiments. The label-free determination of the unknown antibody concentrations was performed using two related kinetic analyses. From these results, the current array assay sensitivity is 250 ng ml^-1 with an accuracy of 15% using an 8-min kinetic measurement and a 16-spot averaged assay.     

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.     

Bioluminescent assay of NADPH-dependent isocitrate dehydrogenase and its substrates and cofactors

A bioluminescent assay for NADPH-dependent isocitrate dehydrogenase and for its substrates and cofactors was developed. The method is based on continuous NADPH monitoring in the reaction. The linear range of the assay for the enzyme activity is from 0.05 U/liter to 30 U/liter. It is about 300 times more sensitive than the corresponding spectrophotometric assay at 340 nm. Good correlation exists between both assays. Isocitrate, NADP, manganese, and magnesium can be measured at picomole levels. The applicability of the assays to serum analysis is discussed.     

On improved calibrations of unknowns in a system of quality-controlled assays

Two new estimators for calibrating unknowns from dose-response curves, in a system of quality-controlled assays, are examined. In contrast with the conventional estimator which uses only the results of the one assay in which the response of the unknown dose is measured, the new estimators also utilize the results of all other assays through the replications of the control samples in the system. The first estimator is based on maximizing the likelihood of the given system (with respect to the different dose-response parameters, the levels of the control samples and the levels of the unknowns) when response errors are normally distributed. The second estimator is a regression-like estimator obtained by subtracting from the conventional estimator its estimated regression on the deviation of the calibrated control levels in the given assay from their average values in the system. Evaluations of the reductions in bias and variance attained by the new estimators show when substantial reductions in mean square error can be expected. The new estimators are illustrated with a system of 22 hFSH radioimmunoassays.     

Logistic Proliferation of Cells in Scratch Assays is Delayed

Scratch assays are used to study how a population of cells re-colonises a vacant region on a two-dimensional substrate after a cell monolayer is scratched. These experiments are used in many applications including drug design for the treatment of cancer and chronic wounds. To provide insights into the mechanisms that drive scratch assays, solutions of continuum reaction–diffusion models have been calibrated to data from scratch assays. These models typically include a logistic source term to describe carrying capacity-limited proliferation; however, the choice of using a logistic source term is often made without examining whether it is valid. Here we study the proliferation of PC-3 prostate cancer cells in a scratch assay. All experimental results for the scratch assay are compared with equivalent results from a proliferation assay where the cell monolayer is not scratched. Visual inspection of the time evolution of the cell density away from the location of the scratch reveals a series of sigmoid curves that could be naively calibrated to the solution of the logistic growth model. However, careful analysis of the per capita growth rate as a function of density reveals several key differences between the proliferation of cells in scratch and proliferation assays. Our findings suggest that the logistic growth model is valid for the entire duration of the proliferation assay. On the other hand, guided by data, we suggest that there are two phases of proliferation in a scratch assay; at short time, we have a disturbance phase where proliferation is not logistic, and this is followed by a growth phase where proliferation appears to be logistic. These two phases are observed across a large number of experiments performed at different initial cell densities. Overall our study shows that simply calibrating the solution of a continuum model to a scratch assay might produce misleading parameter estimates, and this issue can be resolved by making a distinction between the disturbance and growth phases. Repeating our procedure for other scratch assays will provide insight into the roles of the disturbance and growth phases for different cell lines and scratch assays performed on different substrates.     

Assay for beta-ureidopropionase by high-performance liquid chromatography

A sensitive assay for beta-ureidopropionase based on derivatization of the reaction product beta-alanine with phenylisothiocyanate has been developed. Purification of the resulting phenylthiocarbamoyl-beta-alanine is achieved on a LiChrospher 100 C18 reversed-phase high-performance liquid chromatography column using an isocratic elution system. Phenylthiocarbamoyl-beta-alanine is detected by its absorbance at 245 nm and quantitated by automatic peak integration referring to a calibration curve. This technique offers a high degree of sensitivity as beta-alanine quantities in the picomole range can be identified. N-Carbamoyl-beta-alanine, the natural substrate of beta-ureidopropionase, does not interfere with the described assay system. The enzymatic reaction is linear for an incubation time of 45 min with enzyme concentrations of 3.2 micrograms/ml.     

Calibration of Quantitative Histochemical Methods: Estimation of Glycogen Content of Muscle Fibers Using the PAS Reaction

A fairly simple method for calibrating microdensitometric histochemical assays is described. The method is based on paired biochemical and histochemical assays on single freeze-dried skeletal muscle fibers which differ widely in their properties. As an example, the method is applied to investigate the validity of the periodic acid-Schiff (PAS) reaction for the microdensitometric estimation of glycogen content. Some problems that may interfere with the calibration are discussed.     

A nonradioactive dot-blot assay for protein tyrosine kinase activity

A new procedure for the assay of protein tyrosine kinase, based on the detection of phosphorylated tyrosyl residues by using monoclonal antibodies to phosphotyrosine, is described. After incubation of a protein tyrosine kinase sample with the substrates poly-(GluNa,Tyr)4:1 and unlabeled ATP an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining procedure. The signal is quantified by densitometry. The assay is linear with time and is quantitative in a wide range of sample protein concentrations. Its sensitivity allows the kinetic characterization of protein tyrosine kinases at low substrate concentrations, whereas on the other hand the avoidance of radioactivity enables the use of high ATP concentrations as well. Protein tyrosine kinase activities of human breast carcinomas and normal breast tissues measured with this method correlated well with the conventional assay, in which the incorporation of [32P]phosphate is measured by TCA precipitation and liquid scintillation counting. Compared to the latter, the new assay is at least as sensitive and accurate and harbors the advantage of the avoidance of radioactivity, thus enabling one to perform a large number of protein tyrosine kinase assays simultaneously.     

Exponential model describing methane production kinetics in batch anaerobic digestion: a tool for evaluation of biochemical methane potential assays

Biochemical methane potential assays, usually run in batch mode, are performed by numerous laboratories to characterize the anaerobic degradability of biogas substrates such as energy crops, agricultural residues, and organic wastes. Unfortunately, the data obtained from these assays lacks common, universal bases for comparison, because standard protocols did not diffuse to the entire scientific community. Results are usually provided as final values of the methane yields of substrates. However, methane production curves generated in these assays also provide useful information about substrate degradation kinetics, which is rarely exploited. A basic understanding of the kinetics of the biogas process may be a first step towards a convergence of the assay methodologies on an international level. Following this assumption, a modeling toolbox containing an exponential model adjusted with a simple data-fitting method has been developed. This model should allow (a) quality control of the assays according to the goodness of fit of the model onto data series generated from the digestion of standard substrates, (b) interpretation of substrate degradation kinetics, and (c) estimate of the ultimate methane yield at infinite time. The exponential model is based on two assumptions: (a) the biogas process is a two-step reaction yielding VFA as intermediate products, and methane as the final product, and (b) the digestible substrate can be divided into a rapidly degradable and a slowly degradable fraction.     

Playing chicken (Gallus gallus): methodological inconsistencies of molecular divergence date estimates due to secondary calibration points

For any given taxonomic divergence event, one may find in the literature a wide range of time estimates. Many factors contribute to the variation in molecular date estimates for the same evolutionary event. High on the list is the choice of calibration points for converting genetic distances into evolutionary rates and, subsequently, into dates of divergence. In this study, we investigate one critical source of error in estimating divergence times, i.e. the use of secondary calibration points, which are divergence time estimates that have been derived from one molecular dataset on the basis of a primary external calibration point, and which are used again independently of the original external calibration point on a second dataset. Unless particular care is exercised, this practice leads to internal inconsistencies, and the inferred dates of divergence are by necessity unreliable. We present a consistency test for assessing the reliability of divergence time estimates based on secondary calibration points. As a case study, we examine recent estimates of divergence times among phyla and kingdoms based on multiple nuclear protein-coding genes, and show that they fail the consistency test.     

An ultrasensitive enzymatic method for measuring mevalonic acid in serum

We have developed a simple, precise, and ultrasensitive enzymatic method for measuring serum mevalonic acid (MVA) concentration, which is thought to be a good indicator of the in vivo cholesterol biosynthesis rate. This assay is based on an enzyme cycling reaction and makes use of HMG-CoA reductase (HMGR), thio-NAD, NADH, and CoA. MVA participates in the HMGR cycling reaction, and its level is measured based on the production of thio-NADH, which is determined from the change in absorbance at 405 nm. To achieve high specificity, we used mevalonate kinase (MVK) in addition to HMGR. Only substrates able to participate in both the HMGR cycling reaction and the MVK reaction are measured as MVA. The detection limit for MVA is 0.4 ng/ml (2.7 nmol/l), and the calibration curve for MVA is linear up to 44 ng/ml (300 nmol/l). Regression analysis with 40 serum samples showed the accuracy of quantifying MVA with this enzymatic assay to be comparable to that using LC-MS/MS (correlation: y = 0.83x + 0.24; r = 0.97). This procedure is simple, precise, and robust. It is also rapid and has a high throughput, making it potentially useful for clinical applications.     

Growth hormone determination in children using an immunofunctional assay in comparison to conventional assays

Conventional assays for determination of growth hormone (GH) in serum measure immunoreactive molecules of a blood sample. The immunofunctional assay (IFA), on the other hand, is able to determine biologically active molecules. In our study, we evaluated GH determination in children with IFA to compare these data with clinical reliable data of conventional assay systems, since there is only insufficient data concerning the clinical use of IFA in children. The comparison of GH determinations by IFA and two immunoradiometric assays showed different results for the same serum sample. Peak and trough concentration levels determined by these three different assays were always measured at the same time, but absolute GH concentration levels varied. Statistic analysis verified a linear regression of our data and allowed a conversion of data measured by IFA to predict values of the other assays used and vice versa. The traditional cut-off level for the diagnosis of GH deficiency of 10 ng/ml was based originally on results of polyclonal radioimmunoassays. This internationally applied cut-off level has been converted based on the regression analysis for prediction of this study and we found the 95% confidence interval on the mean measurements by IFA to be between 3.11 and 3.28 ng/ml.