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At least 127 records · Page 7Linked to original sources

PubMed2001

Demonstrative validation study employing a packed column pressurized fluid chromatography method that provides assay, achiral impurities, chiral impurity, and IR identity testing for a drug substance.

Assay development, assay validation, and documentation are reported here for a single packed column pressurized fluid chromatographic/ultraviolet (UV) method that provides: (1) simultaneous detection and quantification for the chiral drug, the chiral impurity and seven achiral impurities; and (2) a Fourier transform infrared (FT-IR) spectrometric identification test result for the Searle drug substance sample, xemilofiban. The separation is achieved in less than 30 min with three columns in tandem and a gradient of CO2-CH3OH. The post-column flow is split between UV (assay) and FT-IR (identification). Precision and accuracy are consistent within figures of merit obtained by liquid chromatographic-ultraviolet assays on analogous drug substances. The reported procedure combines three typical drug substance tests into one test (e.g. chiral impurities, achiral impurities, and infrared identification).

Chromatography, Liquid↗

PubMed2002

A homogeneous caspase-3 activity assay using HTRF technology.

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

Biological Assay↗

PubMed2005

Probing the primary screening efficiency by multiple replicate testing: a quantitative analysis of hit confirmation and false screening results of a biochemical assay.

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

DNA-Binding Proteins↗

PubMed2006

An ounce of prevention on a budget: a nonprofit approach to developing vaccines against neglected diseases.

This article provides a perspective on vaccine development for neglected tropical diseases in the nonprofit setting, with particular emphasis on recombinant protein vaccines. The Human Hookworm Vaccine Initiative is discussed as a model product development public-private partnership, and the major challenges are covered that accompany antigen selection, gene cloning, fermentation and purification process development, assay development, vaccine formulation and testing and clinical evaluation for those developing vaccines, especially against neglected tropical diseases, in the nonprofit sector. Throughout this perspective, special emphasis is placed on the growing promise that product development public-private partnerships hold for developing vaccines for the world's poorest people.

Budgets↗

PubMedDate not supplied

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.

Animals↗

PubMedDate not supplied

Fentanyl receptor assay. I. Development of a radioreceptor assay for analysis of fentanyl and fentanyl analogs in urine.

Radioreceptor binding assays may be useful methods for determining the plasma and urine concentration of drugs. We have evaluated the possibility of employing CNS receptors in an assay to measure fentanyl and fentanyl-like drugs. This in vitro assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors in membrane preparations of rat forebrain. The binding is stereospecific, reversible, and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Naloxonazine, which selectively binds to mu 1-opioid site, competed with [3H]fentanyl for its high affinity binding site. Morphine and hydromorphone competed with [3H]fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of [3H]fentanyl. Many other commonly abused drugs did not displace [3H]fentanyl from the opiod receptors. Urine samples from animals injected with fentanyl were evaluated by other analytical techniques, including radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/MS), and results were compared to those from the radioreceptor assay. Urinary analysis of fentanyl showed a good correlation between all three methods.

Animals↗

PubMed2001

Progress in the use of biological assays during the development of biotechnology products.

The complexity of the structure and function of many biotechnology derived products necessitates a wide range of analytical procedures to adequately characterize the product. In-depth characterization is required for the assessment of several criteria vital to the success of product development such as consistency, purity, stability, and potency. More recently, the concern over the immunogenicity of biologics has increased the need to develop assays to detect neutralizing anti-product antibodies. Although many physicochemical tests are available to characterize the structure of a protein and detect the presence of contaminants, they provide little, if any, information regarding biological potency or the neutralizing capacity of antibody responses in immunogenicity studies. There is a continual need to refine biological assays to increase their accuracy and reproducibility, in particular to replace in vivo bioassays with appropriate in vitro assays. There have also been several recent technological developments that could lead to more rapid and reproducible bioassays.

Allergy and Immunology↗

PubMed2005

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine.

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC-MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5-400 ng/mL on PE Sciex API 3000 LC-MS/MS system. The assay intraday accuracy and precision were 92.1-104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299-307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739-752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.

Adsorption↗

PubMed2004

Development of a novel internal positive control for Taqman based assays.

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.

False Negative Reactions↗

PubMed2005

Differential reactivity of anti-primate and anti-human secondary antibodies against human and monkey immunoglobulins: implications for determining the sensitivity of immunogenicity assays.

Development of immunogenicity assays for assessment of human antibodies to therapeutic proteins requires a quantitative determination of assay sensitivity. In the absence of true human positive controls, this is usually accomplished by utilizing affinity-purified antibodies from non-human primates or monoclonal antibodies. In the former case, it is generally considered that non-human primate antibodies will be recognized equally to human antibodies by secondary anti-human immunoglobulin reagents used in immunogenicity assays. We present results here demonstrating that this is not the case. In reality, anti-human immunoglobulin secondary antibodies do not recognize primate immunoglobulins as well as human immunoglobulins. As a result, the use of affinity purified primate antibodies to determine the sensitivity of an immunogenicity assay will likely result in the true sensitivity of the assay being underestimated.

Animals↗

PubMed2004

Miniaturized, ultra-high throughput screening of tyrosine kinases using homogeneous, competitive fluorescence immunoassays.

Assay miniaturization and the implementation of high-density 1,536-microwell screening increase the speed and efficiency of screening and lead discovery. To serve this need, a platform of miniaturizable assay technologies has been assembled for specific biological targets. This platform will enable initiation and completion of uHTS screens in a straightforward and expeditious manner. Although faster primary screening does contribute to a reduction in timelines, the process of assay development can become a bottleneck. Assay technologies that do not require the use of target-specific reagents can reduce the time necessary for assay development. Assays that measure inhibition of tyrosine kinases can be configured in a competitive format where only the enzyme itself is specific to the assay. In this context, several technologies, including time-resolved fluorometry (also known as DELFIA), time-resolved fluorescence resonance energy transfer (also known as LANCE( trade mark )), fluorescence polarization, enzyme fragmentation complementation assay, and confocal laser scanning imaging, were examined. Quality parameters such as assay reproducibility, signal:background ratio, Z factor, and assay sensitivity were compared. Additionally, the relative merits of each of these technologies are assessed in terms of assay miniaturization, ease of development, ultimate screening capability, efficiency, and cost.

Amino Acid Sequence↗

PubMed1999

Development of a PCR assay for rapid detection of enterococci.

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.

Bacterial Typing Techniques↗

PubMed1984

Development of assays for drugs of abuse.

The treatment and prevention of drug abuse has required the development of assays for biological markers to assess an individual's adherence to a treatment regimen or to the prescribed abstinence of drug use. Generally, this requires assays sensitive enough to detect a few nanograms of a particular metabolite per milliliter of urine. Most frequently, the identify of the drug or combination of drugs is not known. This placed a large responsibility on the development of reliable methods. To accomplish this mission, the National Institute on Drug Abuse funded a multiyear, multiple-contract program. This program included the synthesis of reference drugs, their metabolites, and labeled derivatives; the development of several new analytical methods, especially using gas chromatography/mass spectrometry and immunoassay techniques; the establishment of analytical service facilities; animal and human pharmacokinetic studies; and laboratory proficiency testing. Many of the assays are now commercially available and widely used. Assay development requires the consideration of a number of factors, namely, specificity, sensitivity, time, simplicity, and cost. These factors are illustrated in a number of examples.

Gas Chromatography-Mass Spectrometry↗

PubMed1976

Enzymic methods for estimation of the somatic cell count in bovine milk. 1. Development of assay techniques and a study of their usefulness in evaluating the somatic cell content of milk.

Assay procedures were developed for a number of enzymes in milk which apparently originate from leucocytes. The enzymes studied were acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, arylsulphatase, alpha-mannosidase, and catalase. Quarter-milk samples were analysed for enzyme activity and results compared with the electronic cell count and the Wisconsin Mastitis Test. All enzymes measured except acid phosphatase and alpha-mannosidase showed good correlation with the electronic cell count. Of the other 4 enzymes tested, beta-glucuronidase and arylsulphatase were unsuitable as diagnostic aids owing to the lengthy incubation periods required in their assay procedures. The assay of catalase, which involved the measurement of the initial rate of release of O2 using an O2 analyser apparatus, was rapid, sensitive and reasonably reliable, if fresh milk samples were used. The assay procedure for N-acetyl-beta-D-glucosaminidase was considered to be the most reliable, simple and rapid enzymic method for estimating the number of somatic cells in milk.

Acetylglucosaminidase↗

PubMed1997

A laboratorian's perspective on evaluation and implementation of new laboratory tests.

New assay development should be directed toward answering fundamental clinical questions. Caveats that must be considered before initiating assay development projects are: New assays should allow the clinician to interact with and treat a patient more effectively, thereby improving medical outcome; and new assays should facilitate recapture of system resources, enabling cost savings or reinvestment of resources. Defining the clinical questions and consideration of the caveats permit a means of prioritizing assay development activities. Laboratorians are faced with evaluating several types of development activities that lead to assay implementation in routine clinical testing. Assays can be prioritized for up-grading to newer cost-effective technologies, provided the changes maintain or improve analytical and clinical performance. Predicting which research assay will have future value is difficult when clinical performance is not fully validated. However, such assay development has the greatest potential for changing the delivery of healthcare by a clinician.

Biomarkers↗

PubMed1983

Immunoreactivity of human parathyroid hormone (28-48): attempt to develop an assay for intact human parathyroid hormone.

The postsecretory catabolism of parathyroid hormone (PTH) is known to involve proteolytic cleavages in the 28-48 region. In an effort to develop assays that would detect the intact human PTH molecule but not its catabolic products, I have developed radioimmunoassays specific for the 28-48, or cleavage, region. A synthetic fragment of the human PTH molecule, substituted with tyrosine, [27Tyr]hPTH-(27-48), was radioiodinated to serve as tracer. Four of four high titer antisera against native human PTH bound significant amounts of this tracer. Binding of this radioligand was inhibited by unlabeled synthetic human PTH-(28-48) or native human PTH-(1-84) but not by the following synthetic hormone fragments which resemble those thought to be formed in vivo: hPTH-(1-34), hPTH-(44-68), or hPTH-(53-84). The most sensitive assay would detect about 20 fmol of hPTH-(1-84) or 10 fmol of hPTH-(28-48). The bovine PTH 28-48 region crossreacted poorly with the human 28-48 region. Neither bovine PTH-(1-84) nor PTH-(28-48) would inhibit the binding of the labeled human PTH 27-48 peptide to the antisera against native human PTH, nor would the labeled human 27-48 peptide bind to any of nine antisera against bovine PTH. These results confirm that the steric configuration of the 28-48 region of PTH differs greatly between human and bovine PTH. The use of assays specific for the 28-48 region of hPTH appears to be valid approach to the problem of measuring the intact hormone. Improvements in sensitivity may allow detection of intact PTH in human serum.

Animals↗

PubMed1995

Investigation of sheep-associated malignant catarrhal fever virus infection in ruminants by PCR and competitive inhibition enzyme-linked immunosorbent assay.

Development of control measures for the gammaherpesviral disease of cattle known as sheep-associated malignant catarrhal fever (SA-MCF) has been hampered by a lack of accurate diagnostic tests either for the causative virus or for antibody against that virus. A recently developed competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) for the detection of antibody to malignant catarrhal fever (MCF) virus (MCFV) in ruminants based on a monoclonal antibody to a widely conserved epitope of MCFV (H. Li, D. T. Shen, D. P. Knowles, J. R. Gorham, and T. B. Crawford, J. Clin. Microbiol. 32:1674-1679, 1994) and a PCR assay based on previously reported primers (S. I. F. Baxter, I. Pow, A. Bridgen, and H. W. Reid, Arch. Virol. 132:145-159, 1993) were used to detect anti-MCFV antibody and SA-MCFV DNA in sheep and other ruminants. The PCR amplified a specific 238-bp SA-MCFV genomic DNA fragment from peripheral blood lymphocytes of adult sheep and other ruminants with clinical MCF. Of 144 samples from randomly selected healthy adult sheep, 143 (99%) were positive by PCR and 136 (94%) were positive by CI-ELISA. The agreement between the two assays exceeded 95%. Of nine samples collected from cattle and deer with clinical MCF of apparent sheep origin, seven were CI-ELISA positive and all 9 were PCR positive. Among 59 serum samples from presuckling lambs, none contained antibody detectable by CI-ELISA. After suckling, maternal anti-MCFV antibody was detectable for about 10 +/- 3 weeks. Although all colostrum and milk samples from infected ewes were strongly PCR positive, the appearance of detectable SA-MCFV DNA in lambs was correlated generally with antibody patterns, which suggests that the natural infection event in sheep may not occur during the perinatal period but occurs sometime later in life.

Animals↗

PubMed2003

Development of an automated photometric assay for serum lithium and use of binding equilibrium expressions to optimize results.

BACKGROUND: A spectrophotometric serum lithium assay and relevant reaction equilibria used in the assay development are described. METHODS: The dye, 7-nitro-2,12-dicarboxyl-16,17-dihydro-5H,15H-dibenzo[b,i] [1,11,4,5,7,8]dioxatetraazacyclotetradecine reacts rapidly with Li(+) in alkaline water/dimethylsulfoxide with an absorbance change. Sodium binds weakly, thus presenting potential inaccuracy from varying endogenous sodium. To minimize this problem, excess sodium ion was added from reagents. This diminishes the signal from lithium binding, thus lowering sensitivity, requiring selection of reagent sodium concentration for proper balance of performance characteristics. Absorptivities and binding constants were determined. Description of competing equilibria by an explicit cubic equation was used to predict sensitivity and effects of varying endogenous Na(+) concentration as functions of added NaCl. RESULTS: Concentration-absorbance data were satisfactorily predicted by the cubic equation, equilibrium constants, and absorptivities. Li(+) binds 300 times more tightly than Na(+). Interference was minimized, while sufficient sensitivity for needed precision was retained. A full evaluation is summarized separately in this issue. CONCLUSIONS: Explicit description of the competitive binding was successfully used to develop an automated liquid reagent assay for Li(+) with acceptable accuracy and precision.

Automation↗