Position Of Label Research Articles

Octanoate and Nonaoate Oxidation Increases 50–80% Over the First Two Days of Life in Piglet Triceps Brachii and Gracilis Muscle Strips ,

An in vitro muscle strip incubation system was developed to measure the rate of catabolism of 1 mmol/L [1-14C]octanoate, 1 mmol/L [1-14C]nonanoate, 1 mmol/L [9-14C]nonanoate, and 10 mmol/L [U-14C]glucose by measuring the recovery of14CO2. Muscle strips (13 mm × 1.5 mm, ~50 mg) were isolated from triceps brachii and gracilis muscles of newborn and 2-d-old, small (<950 g) and large (>1450 g) piglets. The position of the14C label in the substrate affected the rate and amount of recovery in14CO2. Therefore, comparisons were made between age groups (0 vs. 2 d old) within substrates but limited across substrates to comparisons of [1-14C]–labeled fatty acids. The medium-chain fatty acid (MCFA) oxidation rates [pmol/(h · mg)] in muscle strips isolated from piglets from the 2 weight groups (<950 and >1450 g) did not differ (P> 0.99), there was a trend towards a difference between triceps brachii and gracilis muscle (P= 0.09; data not shown), and there were no significant interactions involving pig weight or muscle type; therefore, results were pooled across these factors. During the first 2 d of life, MCFA oxidation [pmol/(h · · mg muscle strip)] increased (P< 0.05) 50–80%, but the glucose oxidation rate did not change (P> 0.82). By d 2, the oxidation rate of nonanoate as represented by the one carbon was 25% greater than for octanoate (P< 0.05). The conversion of [9-14C]nonanoate to14CO2indicated that muscle had the capacity to oxidize the propionyl-CoA produced by β-oxidation of nonanoate and that odd-chain C-9 MCFA provided anabolic carbon to the citric acid cycle.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

Calculation and Mitigation of Isotopic Interferences in Liquid Chromatography–Mass Spectrometry/Mass Spectrometry Assays and Its Application in Supporting Microdose Absolute Bioavailability Studies

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

The effects of changes in the order of verbal labels and numerical values on children's scores on attitude and rating scales

Research with adults has shown that variations in verbal labels and numerical scale values on rating scales can affect the responses given. However, few studies have been conducted with children. The study aimed to examine potential differences in children's responses to Likert‐type rating scales according to their anchor points and scale direction and to see whether or not such differences were stable over time. A total of 130 British children, aged 9 to 11 years, completed six sets of Likert‐type rating scales, presented in four different ways varying the position of positive labels and numerical values. The results showed, both initially and 8–12 weeks later, that presenting a positive label or a high score on the left of a scale led to significantly higher mean scores than did the other variations. These findings indicate that different arrangements of rating scales can produce different results, which has clear implications for the administration of scales with children.

Aptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates

This study presents and discusses the application of Cy3‐labeled aptamers (where Cy3 is indocarbocyanine) directed against the his‐tag (where his is histidine) for the detection of his‐tagged proteins on microarrays in a so‐called reverse phase assay. These types of assays are widely used tools in protein microarray technology. Up to now antibodies are usually applied as detection molecules. Here, two different spotting techniques, contact and noncontact spotting, as well as different types of slides, aldehyde‐modified glass slides and nitrocellulose membrane coated slides, were examined and compared. Through this study, we validated the importance of a high protein‐binding capacity of the microarray, and the labeling position of the fluorophore within the aptamer. Purified his‐tagged PFEI (Pseudomonas fluorescence esterase I) was used as a model system. Concentrations of PFEI‐his as low as 30 nM were detectable. These results demonstrate the applicability of aptamers as stable detection molecules in protein assays. Additionally, the reverse phase assay was found to be suitable for the detection of PFEI‐his in cell lysates. This might be of further interest in monitoring of protein production and purification processes.

Correction to Significance of Xenobiotic Metabolism for Bioaccumulation Kinetics of Organic Chemicals in Gammarus pulex

The authors regret that in our article1 the data for 2,4-dichlorophenol (CAS 120-83-2) need to be corrected. In Table 1 the corrected BAF of 2,4-dichlorophenol is 38 L/kgwet weight, the corrected MEFM1 (2,4-dichlorophenol-sulfate) is 1935 L/kgwet weight, and the corrected MEFM2 is 129 L/kgwet weight. In Table 2 the corrected model parameters (with 95% confidence intervals) for 2,4-dichlorophenol are 6815 (0; 62871) L × kg–1 × d–1 for kin_parent, 156 (0; 1594) d–1 for kout_parent, 21.27 (0; 48.6) d–1 for kmet1, 0.415 (0.34; 0.49) d–1for kloss_met1, 3.627 (0; 9.8) d–1 for kmet2, and 1.063 (0; 2.69) for kloss_met2. The Supporting Information is also updated with the corrected raw data for the biotransformation modeling and Figure ​Figure11 shows the corrected time course of bioaccumulation, biotransformation, and elimination kinetics for 2,4-dichlorophenol and its metabolites (top part of Figure 3 in original article). Figure 1 Molecular structures, label positions, exposure concentration (left), bioaccumulation, biotransformation, and elimination kinetics (right, transfer to fresh media indicated by dashed line), and model structure (middle) for 2,4-dichlorophenol. The corrected values do not change any conclusion in our article because the relation of BAF to MEFs for 2,4-dichlorophenol did not change and the compound had been excluded from the comparison with the study based on total 14C measurements. Note however, that the corrected BAF and MEF values for 2,4-dichlorophenol are higher than those originally published.

Evolutionary Computation for Label Layout on Unused Space of Stacked Graphs

Placing numerous objects and their corresponding labels in the stacked graph visualization is a challenging problem. In the stacked graph, different combinations of initial parameters and filtering effects yield views with hidden information, illegible labels, and unused space. The result is a tool that does not take advantage on the unused space to reveal information to the user for further investigation. We present an automatic method for label layout on the unused space in a stacked graph. An evolutionary computation (EC) is used to optimize the best label position according to legibility requirements, as well as requirements for keeping semantic relationships between labels and their representative visual objects. A number of EC experiments, as well as a usability study on label legibility, show that our proposed solution looks promising, as compared to the traditional solutions.

Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid–protein nanodomains, termed “rafts”. In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids (“raft lipids”, e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.

Click Strategies for Single-Molecule Protein Fluorescence

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Ultrafast Site-Specific Fluorescence Quenching of 2-Aminopurine in a DNA Hairpin Studied by Femtosecond Down-Conversion

The ΔP(-)PBS analog of the DNA primary binding sequence (PBS) of the HIV-1 genome labeled at different positions by 2-aminopurine (2-AP) is investigated by a novel femtosecond fluorescence down-conversion experiment with 0.3-ps time resolution. The high signal-to-noise ratio of the fluorescence kinetics makes it possible to reveal four distinct decay times ranging from 0.8 ps to 2-3 ns for all the three labeling positions. This suggests the existence of at least four different quenching conformations of 2-AP with its nearest neighbors, and underscores the structural heterogeneity of the loop region of ΔP(-)PBS. Sub-5-ps components are found and attributed to stacking interactions of 2-AP with the flanking guanine (G) side chains, consistent with the NMR structure of ΔP(-)PBS. The observation of a significant increase of their total amplitude when 2-AP is positioned close to the rigid 3'-half of the G-rich stem gives further support to this assignment. Only a minor portion of conformations involves slow nanosecond collisional quenching.

α-Synuclein Monomer Conformations Studied by in Silico and in Vitro FRET

The pathological aggregation of α-synuclein (AS), a 140-aa presynaptic protein, is cytotoxic to motor neurons in the brain and thus constitutes a major factor in the etiology of Parkinson's disease (PD). AS is intrinsically unstructured and adopts no preferred fold in solution. Since low molecular weight oligomers of AS are considered to be the most toxic species in PD it is important to study the conformers of monomeric AS. In our current investigation we combine classical molecular dynamics (MD) simulations with experimental FRET measurements to determine distance relations in the N-terminus and the NAC region of AS. With extended MD simulations (∼ 90μs) we first generated ensemble structures of monomeric AS molecules. From these trajectories snapshots were extracted and the appropriate amino acids mutated at eight different FRET label positions respectively. The FRET pair consists of a tryptophan as the donor and a coumarine dye attached to a cysteine as the acceptor. QM calculations were carried out to parameterize the coumarine dye for the AMBER 99SB force field and to determine transition dipole moments. From the AS MD simulations with the attached FRET labels instantaneous donor-acceptor distances rDA and values of the orientation factor κ2 can be extracted. Applying Forster's theory energy transfer efficiencies are calculated and compared to the experimental findings. With the directly available values rDA and κ2 we get an improved structural characterization of the disordered AS ensemble.M. Hoefling, N.Lima, D.Haenni, C.A. M.Seidel, B.Schuler, H.Grubmuller, PLoSONE 6(5): e19791. doi:10.1371/journal.pone.0019791.A.Grupi, E.Haas, JMB, 405:1267-1283 (2011).M.J.Roberti, C.W.Bertoncini, R.Klement, E.A.Jares-Erijman, T.M.Jovin, Nature Methods, 4(4):345–351 (2007).

Feasibility of pseudocontinuous arterial spin labeling at 7 T with whole-brain coverage

ObjectWe studied the feasibility of pseudocontinuous arterial spin labeling (pCASL) at 7 T.Materials and methodsSimulations were performed to find the optimal labeling parameters for pCASL, with particular attention to the maximum-allowed specific absorption rate (SAR). Subsequently, pCASL experiments (four volunteers) were performed to find the B1 efficiency at the labeling position with and without high-permittivity pads placed around the head, and to study the optimal labeling duration (four separate volunteers). Finally, feasibility of whole-brain pCASL imaging was tested.ResultsSimulations showed that a lower B1 efficiency should be compensated by a lower effective flip angle of the labeling, a moderately shorter labeling duration, and a longer repetition time. B1 efficiency in the internal carotid arteries just below the carotid siphon was approximately 55% and 35% with and without high-permittivity pads, respectively. In vivo experiments showed an optimal labeling duration of 1,500 ms, although longer labeling durations up to 2,500 ms resulted in similar signal-to-noise efficiency. Whole-brain pCASL imaging was demonstrated in a single volunteer.ConclusionDespite decreased B1 efficiency, sufficient labeling efficiency can be achieved for whole-brain pCASL at 7 T with high-permittivity pads. However, image quality is still limited compared with 3 T, probably due to imaging instabilities, and further research is needed to elucidate this.

Focus+Context Metro Maps

We introduce a focus+context method to visualize a complicated metro map of a modern city on a small displaying area. The context of our work is with regard the popularity of mobile devices. The best route to the destination, which can be obtained from the arrival time of trains, is highlighted. The stations on the route enjoy larger spaces, whereas the other stations are rendered smaller and closer to fit the whole map into a screen. To simplify the navigation and route planning for visitors, we formulate various map characteristics such as octilinear transportation lines and regular station distances into energy terms. We then solve for the optimal layout in a least squares sense. In addition, we label the names of stations that are on the route of a passenger according to human preferences, occlusions, and consistencies of label positions using the graph cuts method. Our system achieves real-time performance by being able to report instant information because of the carefully designed energy terms. We apply our method to layout a number of metro maps and show the results and timing statistics to demonstrate the feasibility of our technique.

Location, Location, Location: Eye-Tracking Evidence that Consumers Preferentially View Prominently Positioned Nutrition Information

Background Nutrition Facts labels can keep consumers better informed about their diets' nutritional composition, however, consumers currently do not understand these labels well or use them often. Thus, modifying existing labels may benefit public health. Objective The present study tracked the visual attention of individuals making simulated food-purchasing decisions to assess Nutrition Facts label viewing. Primary research questions were how self-reported viewing of Nutrition Facts labels and their components relates to measured viewing and whether locations of labels and specific label components relate to viewing. Design The study involved a simulated grocery shopping exercise conducted on a computer equipped with an eye-tracking camera. A post-task survey assessed self-reported nutrition information viewing, health behaviors, and demographics. Subjects/setting Individuals 18 years old and older and capable of reading English words on a computer (n=203) completed the 1-hour protocol at the University of Minnesota during Spring 2010. Statistical analyses Primary analyses included χ 2, analysis of variance, and t tests comparing self-reported and measured viewing of label components in different presentation configurations. Results Self-reported viewing of Nutrition Facts label components was higher than objectively measured viewing. Label components at the top of the label were viewed more than those at the bottom, and labels positioned in the center of the screen were viewed more than those located on the sides. Conclusions Nutrition Facts label position within a viewing area and position of specific components on a label relate to viewing. Eye tracking is a valuable technology for evaluating consumers' attention to nutrition information, informing nutrition labeling policy (eg, front-of-pack labels), and designing labels that best support healthy dietary decisions.

Evaluation of laser labeling on growth and survival of the freshwater pearl mussel, Hyriopsis cumingii

Laser labeling techniques have been developed for breeding programs in aquatic organisms through this study. This study assessed their use in Hyriopsis cumingii, an important Chinese freshwater pearl mussel. In preliminary studies, H. cumingii juveniles between 55 and 95 days old were labeled by a laser engraving machine. The mortality rate decreased in an age-dependent manner, no differences in growth between treated and unlabeled juveniles at 85 and 95 days of age. Hundred-day-old juveniles were labeled with four different sized labels. The mortality rate and growth inhibition were significantly higher with the largest label size (2.55 cm) compared to the smaller size and control. Four different types of laser intensity were applied to 100-day-old juveniles, but mortality rates and growth inhibition after high current treatments were significantly higher than the lower currents and control. The wing part, center, lower edge, left edge and right edge were labeled in different 100-day-old juveniles. Labeling of the left edge caused higher mortality and growth inhibition than the other sites. No significant differences were found after either side of the shell was labeled compared to the control. All labels remained clear without any deformation after several months. In summary, the optimal strategy for the laser labeling of H. cumingii would be in juveniles >85 days. The label size, laser intensity and positions could affect their growth and survival. There was no difference labeling either side of the shell. This study showed laser labeling is a relatively simple and practical marking method for mussels such as H. cumingii.

Effect of Ferrocene Position on Charge Transfer in ds-DNA Films

Ferrocene-labeled DNA systems where the Fc group is directly attached to deoxyuridine are investigated electrochemically in an effort to elucidate the electron transfer properties of these systems. The rigid linkage between the Fc group and the nucleobase provides control over the position of the Fc group within the ds-DNA and helps to elucidate the effect of the position of the redox label on the electrochemical behaviour of the ds-DNA films. Cyclic voltammetry, electrochemical surface plasmon resonance and scanning electrochemical microscopy measurements show that charge transfer (CT) in such systems is to a large extent controlled by the dynamic properties of surface-bound ds-DNA rather than by the CT through base-pair stacks.

TreeRipper web application: towards a fully automated optical tree recognition software

BackgroundRelationships between species, genes and genomes have been printed as trees for over a century. Whilst this may have been the best format for exchanging and sharing phylogenetic hypotheses during the 20th century, the worldwide web now provides faster and automated ways of transferring and sharing phylogenetic knowledge. However, novel software is needed to defrost these published phylogenies for the 21st century.ResultsTreeRipper is a simple website for the fully-automated recognition of multifurcating phylogenetic trees (http://linnaeus.zoology.gla.ac.uk/~jhughes/treeripper/). The program accepts a range of input image formats (PNG, JPG/JPEG or GIF). The underlying command line c++ program follows a number of cleaning steps to detect lines, remove node labels, patch-up broken lines and corners and detect line edges. The edge contour is then determined to detect the branch length, tip label positions and the topology of the tree. Optical Character Recognition (OCR) is used to convert the tip labels into text with the freely available tesseract-ocr software. 32% of images meeting the prerequisites for TreeRipper were successfully recognised, the largest tree had 115 leaves.ConclusionsDespite the diversity of ways phylogenies have been illustrated making the design of a fully automated tree recognition software difficult, TreeRipper is a step towards automating the digitization of past phylogenies. We also provide a dataset of 100 tree images and associated tree files for training and/or benchmarking future software. TreeRipper is an open source project licensed under the GNU General Public Licence v3.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Experimental and Theoretical Spectroscopic Study of 310-Helical Peptides Using Isotopic Labeling to Evaluate Vibrational Coupling

Coupling between the amide linkages in a peptide or protein is the key physical property that gives vibrational spectra and circular dichroism sensitivity to secondary structures. By use of (13)C isotopic labeling on individual and pairs of amide C═O groups, the amide I band for selected residues was effectively isolated in designed hexa- and octapeptides having dominant 3(10)-helical conformations. The resultant frequency and intensity responses were measured with IR absorption, vibrational circular dichroism (VCD), and Raman spectroscopies and simulated with density functional theory (DFT) based computations. Band fitting the spectral components and correlating the results to the computed coupling between selected labeled positions were used to determine coupling constant signs and to estimate their magnitudes for specific sequences. The observed frequency and intensity patterns, and their variation between IR and VCD with label position in the sequence, follow the theoretical predictions to a large degree, but are complicated by end effects that alter the local force field (FF) for some residues in these short peptides. These FF variations were overestimated in the theoretical models which may be evidence of structural variations not included in the model. By analyzing the simulations with different coupling models, the coupling constants were determined to lie in a range (positive) +3-5 cm(-1) for sequential residues (i,i+1) and with (negative) -3 cm(-1) as an upper bound for alternate ones (i,i+2). The sequential amide coupling for 3(10)-helices is weaker than for α-helices but has the same sign and is larger than and oppositely signed as compared to 3(1)-, or poly-(Pro)(n) type-II, helices.

INFLUENCE OF DIFFERENT HOMOLOGOUS AND HETEROLOGOUS COMBINATIONS OF ANTIBODIES AND ENZYME CONJUGATES OF DEHYDROEPIANDROSTOSTERONE ON THE SENSITIVITY AND SPECIFICITY OF DHEA ELISA

Anti-sera were raised against three immunogen: dehydroepiandrostosterone-17-carboxymethyl-oxime-bovine serum albumin (DHEA-17-CMO-BSA), DHEA-7-CMO-BSA, and dehydroepiandrostosterone-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA). They were evaluated with horseradish peroxidase (HRP)-labeled DHEA-17-CMO, DHEA-7-CMO, DHEA-3-HS enzyme conjugates for their influence on the sensitivity and specificity of ELISA. Of the various combinations, DHEA-3-HS-BSA antiserum along with DHEA-7-CMO-horseradish peroxidase (DHEA-7-CMO-HRP) enzyme conjugate showed no cross-reaction with any of the closely related steroids. All the homologous combinations appeared to be less sensitive due to their low affinity for dehydroepiandrostosterone. Out of six heterologous systems tested, only three combinations, (1) anti-DHEA-17-CMO antiserum and DHEA-7-CMO-horseradish peroxidase, (2) anti-DHEA-7-CMO-antiserum and DHEA-3-HS-horseradish peroxidase, and (2) anti-DHEA-3-HS-antiserum and DHEA-7-CMO-horseradish peroxidase, showed displacement. The former two assays were less specific; the first one showed 15.38% and 16.66% cross-reaction with androstenediol and testosterone, respectively, whereas the second assay showed 30.3%, 22.72%, 111.1%, 62.5%, and 31.25% cross-reaction with DHEA-glucuronide, 16-dihydroxyprogesterone, androstenediol, etiocholon-3-β-ol-17-one, and aldosterone, respectively. The ability of DHEA to displace the DHEA-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the DHEA molecule as well as on the availability of antigenic sites in particular combinations of antibody and DHEA-enzyme conjugates.