Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: optimization and data analysis

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.     

Isolation of Ether-Resistant Enteroviruses from Sewage: Methodology

Experiments were conducted to determine whether polio type 1 (Mahoney and coxsackie A8 viruses adsorb onto cotton fibers of sewer swabs. Negative results were obtained. It has been shown that viruses may exist in sewage as free virus particles or as bound (adsorbed) virus particles. The sewer-swab method of sampling is superior because it filters out the bound virus over several days; when collected, it represents a catch (grab) sample at that particular time which may or may not contain free virus. A simple method for the preparation of sewage inocula for virus isolations is described which samples the bound virus fraction. Only ether-resistant viruses can be isolated, and an ultracentrifuge is not required. By this method, an isolation rate between 60 and 80% of positive sewer swabs can be achieved. Corresponding figures of 84 and 96% were achieved by concentration of sewer-swab eluates with an ultracentrifuge. Quantitative studies showed that the virus concentration in raw sewage can be as high as one infectious particle per 0.5 ml.     

Development and validation of a capillary zone electrophoresis method for the determination of atropine, homatropine and scopolamine in ophthalmic solutions

A capillary zone electrophoresis method is described for the simultaneous determination of atropine, homatropine and scopolamine. Successful results were obtained after optimization of the electrophoretic parameters such as buffer composition and pH. The best separation was achieved using a 100 mM Tris-phosphate running buffer at pH 7. The validation data proved that the method had the requisite selectively, reproducibility and linearity to be used for the assay of these compounds in pharmaceutical formulations. Dosage of the separate drugs in ophthalmic preparations is also presented.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Effects of pressure on the composition density distribution in the ultracentrifuge

The effects of pressure on the composition density distribution in a binary density gradient at sedimentation equilibrium in the analytical ultracentrifuge are rigorously examined. A computer algorithm is described for the necessary iterative computations. The pressure effect is found to be significant in runs where a long column length, high angular velocity, and a salt with a high pressure correction coefficient are simultaneously employed. Such conditions are sometimes encountered in current studies in which high precision is required to measure the compressibility of proteins.     

Chromatographic determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA: a validation study

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.     

A centrifugation method for the isolation of plasmid DNA without the use of an ultracentrifuge

This paper describes the use of a high-performance high-speed centrifuge, a Sorvall RC-28S, for the preparation of plasmid DNA using standard CsCl-ethidium bromide gradients. If the sample is layered into the bottom of the gradient, the plasmid DNA can be banded in 24 hours; this is comparable with the time required for ultracentrifuge separations. Alternatively, plasmids can be banded in a fixed-angle rotor in 36 hours when the sample is mixed throughout the gradient solution.     

ELISA microarray technology as a high-throughput system for cancer biomarker validation

A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. An additional challenge with cancer screening is that most cancers are rare in the general population, meaning that assay specificity must be very high. Otherwise, the number of false positives will be much greater than the number of true positives. Due to these challenges associated with biomarker validation, it is necessary to analyze thousands of samples in order to obtain a clear idea of the utility of a screening assay. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and, thus, has the potential to accelerate validation of protein biomarkers for clinical use. This review will discuss current ELISA microarray technology and potential advances that could help to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.     

Standard guidelines for the publication of telomere qPCR results in evolutionary ecology

Telomere length has been used as a proxy of fitness, aging and lifespan in vertebrates. In the last decade, dozens of articles reporting on telomere dynamics in the fields of ecology and evolution have been published for a wide range of taxa. With this growing interest, it is necessary to ensure the accuracy and reproducibility of telomere length measurement techniques. Real‐time quantitative PCR (qPCR) is routinely applied to measure relative telomere length. However, this technique is highly sensitive to several methodological variables and the optimization of qPCR telomere assays remains highly variable between studies. Therefore, standardized guidelines are required to enable the optimization of robust protocols, and to help in judging the validity of the presented results. This review provides an overview of preanalytical and analytical factors that can lead to qPCR inconsistencies and biases, including: (a) sample type, collection and storage; (b) DNA extraction, storage and quality; (c) qPCR primers, laboratory reagents, and assay conditions; and (d) data analysis. We propose a minimum level of information for publication of qPCR telomere assays in evolutionary ecology considering the methodological pitfalls and sources of error. This review highlights the complexity of the optimization and validation of qPCR for telomere measurement per se, demonstrating the importance of transparency and clarity of reporting methodological details required for reliable, reproducible and comparable qPCR telomere assays. We encourage efforts to implement standardized protocols that ensure the rigour and quality of telomere dynamics studies.     

Screening,library-assisted identification and validated quantification of oral antidiabetics of the sulfonylurea-type in plasma by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

An atmospheric pressure chemical ionization liquid chromatographic-mass spectrometric (APCI-LC-MS) LC-MS assay is presented for fast and reliable screening and identification as well as precise and sensitive quantification of oral antidiabetics of the sulfonylurea-type (OADs) in plasma. It allowed the specific diagnosis of an overdose situation or a Munchausen syndrome caused by ingestion of OADs. After liquid-liquid extraction, the OADs glibenclamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, tolazamide and tolbutamide were separated using fast gradient elution. After screening and identification in the scan mode using our new LC-MS library, the OADs were quantified in the selected-ion mode. The quantification assay was validated according to the criteria established by the Journal of Chromatography B. All validation data were inside the required limits. The assay is part of a general LC-MS procedure for fast screening, identification and quantification of different toxicologically relevant compounds in plasma and has proven to be appropriate for OADs.     

Chromatographic determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA: A validation study

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable.

The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.     

Application of the β-expectation tolerance interval to method validation of the M30 and M65 ELISA cell death biomarker assays

Method validation should focus on demonstrating that an assay is fit for its intended purpose. We have applied the β-expectation tolerance interval - a statistical approach that predicts the accuracy of assay measurements in the future - to the validation of two different cell death biomarker assays, the M30 and M65 ELISAs. A meta-analysis was conducted on a total of 57 different M30 and M65 assays run over a 2 year period. All code utilised in calculations was developed using MATLAB. The optimal fit to the calibration curve for the M30 assay was shown to be a quartic curve which yielded a β-expectation tolerance interval of +20.5% and -23.6% at β=95% over a wide range of QC standards (88-810 U/L). However, such a fit required at least 7 points to avoid problems with over fitting. A linear fit to the M65 calibration curve normally produced a tolerance interval of less than ±20%, however, marked inter-batch variations were evident. Amelioration of batch to batch variations was accomplished by fitting M65 calibration data preferably to a 4-parameter logistic function or a cubic spline. The minimum number of QC replicates and different assays required to produce reliable accuracy profiles was determined. The β-expectation tolerance interval approach has resulted in further optimisation of the M30 and M65 ELISAs as biomarker assays that should translate into greater accuracy in results generated from clinical trials samples.     

New insights for rapid evaluation of bactericidal activity: a semi-automated bioluminescent ATP assay

Aims: A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use. Methods and Results: The bactericidal activity of biocides was quantified using a novel semi‐automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri‐phosphate (ATP) content quantification instead of traditional culture‐based microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN 13727. During the validation, the linearity of the ATP detection system, limit of detection, specificity, sensitivity, relative accuracy and precision (repeatability and reproducibility) were determined. Conclusions: The validation study showed that the new assay evaluates the activity of biocides as well as the EN standard suspension tests, but it allows a large number of test conditions to be efficiently analysed. Significance and Impact of the Study: The new test can therefore be applied to accurately establish the lowest active concentration (MBCs) of disinfectants or antiseptics under simulated practical conditions of use and to compare the susceptibility of a large number of strains and conditions via inactivation curves. This is not possible in any reasonably practicable way with the EN standards considering the time and cost required for each determination.     

Analysis of chemically reacting systems by sedimentation-diffusion equilibrium.

A novel procedure to evaluate equilibrium constants from sedimentation-diffusion equilibrium data of analytical ultracentrifuge runs is proposed. It is shown that, by comparison of a reacting mixture at chemical equilibrium with a non-reacting but equally composed one, the sum of the mean concentrations of the reaction products can immediately be taken from optical absorption or from interferometric measurements. In most but not in all cases the use of stacked double-sector centerpieces is required.     

Assessment of assay precision: a case study of an ELISA for anti-pertussis antibody.

Assay evaluation and validation is essential to ensure that assays are sufficiently specific and provide estimates with sufficient precision for the required purposes. This must be an on-going process, and assays should therefore be designed to permit some degree of both direct and indirect measurement of intra- and inter-assay variation. Quality control procedures may contribute relevant information, but may not be sufficient. Results obtained by two laboratories using as nearly as possible identical reagents and samples in an ELISA for anti-pertussis antibodies are described. These results illustrate aspects of assay performance which may be overlooked once a formal "validation exercise" has been carried out and assays are in routine use. This study also illustrates the information, in addition to that available from in-house studies, which may be provided by appropriately designed inter-laboratory studies, such as the collaborative studies carried out for characterization and calibration of reference materials and standards.     

Optimal design and experimental validation of a modified four-zone SMB process for the separation of a ternary amino acid mixture

For a standard four-zone simulated moving bed (SMB) chromatography, several process modifications have been made in previous studies such that its application scope could be extended to a ternary separation. One of the effective modifications reported was to (1) replace its closed-loop configuration with an open-loop configuration and (2) utilize the extract port for collecting both the intermediate-affinity and the highest-affinity components in regular sequence in every switching period. Most of previous researches on such a modified four-zone SMB (MF-SMB) have been limited to process simulation and optimization. The experimental validation of the MF-SMB process with linear isotherms was attempted in this article for the first time using a ternary amino acid mixture as a model system. First, the intrinsic parameters of three amino acids were estimated from a series of multiple-frontal experiments. The estimated parameter values were then used in the stage of the MF-SMB optimization, which was assisted by an up-to-date genetic algorithm. Based on the optimized conditions, the MF-SMB experiment was conducted and the assay results for product samples verified the attainment of a ternary separation. The experimental purities and concentrations were also found to agree closely with the model predictions.     

Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay

Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP^d) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay’s sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.     

A simple method to measure effective catalase activities: optimization, validation, and application in green coffee

Oxidative metabolism in coffee cherries during maturation appears to be regulated by the timely expression of redox enzymes such as catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO). Among these enzymes, CAT is suspected to contribute significantly in setting the redox status of the healthy cherry and the processed bean. The initial redox status of the green bean might further control the nature and dynamics of reactions induced by roasting and eventually quality aspects of the end product. In this respect, Arabica (Coffea arabica) and Robusta (Coffea canephora) typically differ by their cup coffee flavor profiles. We developed an assay that allowed us to screen numerous green coffee samples for effective CAT activities. The proposed assay, which monitors CAT activities by online oxygen sensing in green coffee crude suspensions incubated with H2O2, seeks to integrate potential effects of endogenous inhibitors and activators. After optimization and validation of the assay, 23 Arabicas, 23 Robustas, and 8 Arabustas were analyzed. Nearly all Arabicas (22 of 23) harbored high CAT activity levels, whereas all Robustas harbored low ones. Arabustas performed like Arabicas of the lower CAT activity range. The traditional spectrophotometric assay did not reveal these specificities. Because of its simplicity, our assay might be valuable for assessing effective CAT activities in various plant tissues.