Biomarkers of dementia: comparison of electrochemiluminescence results and reference ranges with conventional ELISA

We compared the performance of the Meso Scale Diagnostics electrochemiluminescence (MSD) multiplex assay for t-tau and p-tau(231), originally developed for measurement of brain cell extract and tissue cultures, with the established standard method, the Innogenetics ELISA for total and p-tau(181). The methods were also clinically evaluated with 120 samples from our mono center population. The established Innogenetics ELISA procedures have been well optimized to measure patient samples in the normal and pathological range. Compared to the MSD they were superior in the limit of detection for total as well as p-tau. The obtained reference values for our normal controls were in the upper third of the published studies. Innogenetics tau, Innogenetics p-tau(181) and MSD t-tau differentiated the Alzheimer's (n=44) and minimal impairment group (MCI, n=39) from normal controls (n=37), but the MCI group was not statistically different from the normal controls. The MSD multiplex assay measured t-tau adequately but p-tau(231) could not differentiate normal from pathological results in CSF due to the high limit of detection. Both procedures however, have to be further standardized and complemented by adequate internal and external quality control schemes to qualify for routine analysis in a medical laboratory.     

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors

Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC₅₀ 0.7 nM, FMS cell IC₅₀ 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 μg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 μg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Characterisation of grids of point detectors in maximum skin dose measurement in fluoroscopically-guided interventional procedures

PurposePoint detectors are frequently used to measure patient's maximum skin dose (MSD) in fluoroscopically-guided interventional procedures (IP). However, their performance and ability to detect the actual MSD are rarely evaluated. The present study investigates the sampling uncertainty associated with the use of grids of point detectors to measure MSD in IP.MethodChemoembolisation of the liver (CE), percutaneous coronary intervention (PCI) and neuroembolisation (NE) procedures were studied. Spatial dose distributions were measured with XR-RV3 Gafchromic^® films for 176 procedures. These distributions were used to simulate measurements performed using grids of detectors such as thermoluminescence detectors, with detector spacing from 1.4 up to 10 cm.ResultsThe sampling uncertainty was the highest in PCI and NE procedures. With 40 detectors covering the film area (36 cm × 44 cm), the maximum dose would be on average 86% and 63% of the MSD measured with Gafchromic^® films in CE and PCI procedures, respectively. In NE procedures, with 27 detectors covering the film area (14 cm × 35 cm), the maximum dose measured would be on average 82% of the MSD obtained with the Gafchromic^® films.ConclusionThermoluminescence detectors show good energy and dose response in clinical beam qualities. However the poor spatial resolution of such point-like dosimeters may far outweigh their good dosimetric properties. The uncertainty from the sampling procedure should be estimated when point detectors are used in IP because it may lead to strong underestimation of the MSD.     

Performance of high-throughput DNA quantification methods

Background

The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen^® assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses.

Results

The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures.

Conclusion

The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

     

Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed,Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5⁺/6⁺ (BSGP)5⁺/6⁺. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.     

Critical appraisal of four IL-6 immunoassays

Background

Interleukin-6 (IL-6) contributes to numerous inflammatory, metabolic, and physiologic pathways of disease. We evaluated four IL-6 immunoassays in order to identify a reliable assay for studies of metabolic and physical function. Serial plasma samples from intravenous glucose tolerance tests (IVGTTs), with expected rises in IL-6 concentrations, were used to test the face validity of the various assays.

Methods and Findings

IVGTTs, administered to 14 subjects, were performed with a single infusion of glucose (0.3 g/kg body mass) at time zero, a single infusion of insulin (0.025 U/kg body mass) at 20 minutes, and frequent blood collection from time zero to 180 minutes for subsequent Il-6 measurement. The performance metrics of four IL-6 detection methods were compared: Meso Scale Discovery immunoassay (MSD), an Invitrogen Luminex bead-based multiplex panel (LX), an Invitrogen Ultrasensitive Luminex bead-based singleplex assay (ULX), and R&D High Sensitivity ELISA (R&D). IL-6 concentrations measured with MSD, R&D and ULX correlated with each other (Pearson Correlation Coefficients r = 0.47–0.94, p<0.0001) but only ULX correlated (r = 0.31, p = 0.0027) with Invitrogen Luminex. MSD, R&D, and ULX, but not LX, detected increases in IL-6 in response to glucose. All plasma samples were measurable by MSD, while 35%, 1%, and 4.3% of samples were out of range when measured by LX, ULX, and R&D, respectively. Based on representative data from the MSD assay, baseline plasma IL-6 (0.90±0.48 pg/mL) increased significantly as expected by 90 minutes (1.29±0.59 pg/mL, p = 0.049), and continued rising through 3 hours (4.25±3.67 pg/mL, p = 0.0048).

Conclusion

This study established the face validity of IL-6 measurement by MSD, R&D, and ULX but not LX, and the superiority of MSD with respect to dynamic range. Plasma IL-6 concentrations increase in response to glucose and insulin, consistent with both an early glucose-dependent response (detectable at 1–2 hours) and a late insulin-dependent response (detectable after 2 hours).     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

Radioenzymatic microassay for picogram quantities of serotonin or acetylserotonin in biological fluids and tissues

This paper describes several modifications of the original radioenzymatic assay for serotonin which increase the sensitivity of the assay 20-fold as well as enhance its reliability. Using this method serotonin concentrations can be directly measured in biological examples without precleaning the sample. When compared to currently available methods this assay is specific and sensitive to approximately 1 pg of serotonin and can be used to measure serotonin levels in individual brain nuclei or microliter quantities of biological fluids. This assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (<20 cigarettes smoked per day), (3) non-smokers, and (4) vegetarian non-smokers. Samples were evaluated by a gas chromatography/mass selective detector (GC/MSD) method as a reference and using NicCheck I? (DynaGen, Inc.). Colour intensity can range from 0 (no colour) to 14 (deep pink). Qualitative values were assigned as negative (0), low (1-6) and high (7-14). Comparison of the test strip and GC/MSD results showed: (1) 43 (10.75%) false negatives using the criterion of a GC/MSD cotinine level above 200 ng ml^-1 and test strip reading of 0, (2) 31 (7.75%) false positives using the criterion of a GC/MSD cotinine level below 1 ng ml^-1 and a test strip reading of 1 or greater, and (3) no correlation between the test strip and GC/MSD results (r = 0.597, p < 0.05). The fact that the colorimetric reaction is sensitive to many nicotine metabolites and/or heterocyclic amine structures whereas the GC/MSD method measures nicotine and cotinine selectively might explain the false positive results. False negative results were likely to be due to a lack of sensitivity of the test strip.     

Determination of Dopamine-β-hydroxylase Activity in Human Serum Using UHPLC-PDA Detection

Dopamine-β-hydroxylase (DBH, EC 1.14.17.1) is an enzyme with implications in various neuropsychiatric and cardiovascular diseases and is a known drug target. There is a dearth of cost effective and fast method for estimation of activity of this enzyme. A sensitive UHPLC based method for the estimation of DBH activity in human sera samples based on separation of substrate tyramine from the product octopamine in 3 min is described here. In this newly developed protocol, a Solid Phase Extraction (SPE) sample purification step prior to LC separation, selectively removes interferences from the reaction cocktail with almost no additional burden on analyte recovery. The response was found to be linear with an r²?=?0.999. The coefficient of variation for assay precision was <?10% and recovery?>?90%. As a proof of concept, DBH activity in sera from healthy human volunteers (n?=?60) and schizophrenia subjects (n?=?60) were successfully determined using this method. There was a significant decrease in sera DBH activity in subjects affected by schizophrenia (p?<?0.05) as compared to healthy volunteers. This novel assay employing SPE to separate octopamine and tyramine from the cocktail matrix may have implications for categorising subjects into various risk groups for Schizophrenia, Parkinson’s disease as well as in high throughput screening of inhibitors.     

A New Non-radioactive Method for IL-2 Bioassay

An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine (^[3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absor-bance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the ^[3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the ^[3H]thymidine employed in the conventional proliferation assays.     

Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors

Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling regulates the differentiation and function of tumor-associated macrophages (TAMs) that play an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21 showed strong activity (IC₅₀?=?2?nM) against FMS kinase and served as candidate for proof of concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human breast adenocarcinoma cells.     

Validation of an Optimized ELISA for Quantitative Assessment of Epstein-Barr Virus Antibodies from Dried Blood Spots

Our objective was to validate a commercially available ELISA to measure antibody titers against Epstein-Barr virus (EBV) in dried blood spots (DBS) to replace a previously validated assay for DBS that is no longer available. We evaluated the precision, reliability, and stability of the assay for the measurement of EBV antibodies in matched plasma, fingerprick DBS, and venous blood DBS samples from 208 individuals. Effects of hematocrit and DBS sample matrix on EBV antibody determination were also investigated, and the cutoff for seropositivity in DBS was determined. A conversion equation was derived to enable comparison of results generated using this method with the former DBS method. There was a high correlation between plasma and DBS EBV antibody titers (R² = 0.93) with very little bias (?0.07 based on Bland-Altman analysis). The assay showed good linearity and did not appear to be affected by the DBS matrix, and physiological hematocrit levels had no effect on assay performance. There was reasonable agreement between DBS EBV titer estimates obtained using this assay and the previously validated assay (R² = 0.72). The commercially available ELISA assay for EBV antibody titers that we validated for use with DBS will facilitate continued investigation of EBV antibody titers in DBS.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[³²P]pyrophosphate (PP_i) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P_i) concentrations after degradation by inorganic pyrophosphatase of the PP_i released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A₄, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[³²P]PP_i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.     

Heterogeneity of psychophysiological stress responses in fibromyalgia syndrome patients

Dysregulated psychophysiological responses have been observed in patients with fibromyalgia syndrome (FMS), although the results are inconsistent. Surface electromyographic (EMG), systolic and diastolic blood pressure, heart rate (HR), and skin conductance levels (SCLs) were continuously recorded at baseline, and during a series of stress and relaxation tasks in 90 FMS patients and 30 age and sex matched healthy controls (HCs). The patient sample demonstrated lower baseline EMG levels compared to the HCs on all tasks. In contrast, the patients displayed elevated HR and SCL (sympathetic vasomotor and sudomotor indices, respectively) during both stress tasks. A cluster analysis identified four psychophysiological response patterns: 63.3% of HCs showed increased muscle tension and stable cardiovascular responses; 34.8% of FMS patients showed a pattern of increased sympathetic vasomotor reactivity with stable sudomotor and reduced muscular response; 12.2% of FMS patients showed a pattern of increased sympathetic sudomotor reactivity connected with increased sympathetic vasomotor response and reduced muscular response; and, in contrast, 46.7% of FMS patients showed a pattern of parasympathetic vasomotor reactivity and reduced sudomotor as well as muscular response. The identification of low baseline muscle tension in FMS is discrepant with other chronic pain syndromes and suggests that unique psychophysiological features may be associated with FMS. The different psychophysiological response patterns within the patient sample support the heterogeneity of FMS.