Remembrance: NIH scientists develop specific assays for human chorionic gonadotropin.
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Chorionic Gonadotropin↗
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Results for “Assay development”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
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Development of assay system for immunoglobulin production regulatory factors using whole cell cultures of mouse splenocytes.
We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, beta-lactoglobulin, alpha-casein, and beta-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.
Animals↗
Rapid detection of UGT1A1 gene polymorphisms by newly developed Invader assay.
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Antineoplastic Agents, Phytogenic↗
In vitro approaches to evaluate ADMET drug properties.
In vitro assays developed for the evaluation of drug-like properties can accelerate the drug development process. The key assays are those for the evaluation of bioavailability, metabolic stability, drug-drug interaction potential, and toxicity. For bioavailability, the human colon carcinoma derived Caco-2 assay is the most widely used, allowing the evaluation of multiple pathways of intestinal absorption including paracellular uptake, transcellular uptake, and transporter-mediated uptake and efflux. For metabolic stability and drug-drug interactions, human liver microsomes, hepatocytes, and cDNA-expressed microsomes are commonly used, with human hepatocytes representing the most complete system, containing all metabolic enzymes and cofactors at physiological level and an intact plasma membrane to allow the modeling of intracellular drug concentrations. Primary human cells from target organs (e.g., human hepatocytes for human hepatotoxicity) should represent the best experimental system for the evaluation of human drug toxicity. These assays, when applied intelligently with their limitations, should greatly facilitate the selection of drug candidates with a high probability of clinical success.
Biological Availability↗
Development of a saliva sampling method and a rocket immunoelectrophoretic assay for quantification of salivary IgA in the squirrel monkey (Saimiri sciureus).
There has long been the requirement for a marker of well being and chronic stress that is, itself, unaffected by the transient stressors associated with sampling. Work done in the human, the rat and the dog suggests that salivary IgA may fulfil these criteria. This is the first report on the establishment of a saliva sampling technique and immunoelectrophoretic assay for quantification of secretory Immunoglobulin A (s-IgA) in the squirrel monkey (Saimiri sciureus). A safe and effective sampling method is described, as well as a detailed description of an accurate rocket immunoelectrophoretic assay developed for this study. Preliminary results demonstrate a large intra-individual variation, as well as inter-individual variation in s-IgA levels between successive days. This indicates that if stress is associated with decreased s-IgA levels in the squirrel monkey, the usefulness of s-IgA as a stress marker may be limited because analyses of large numbers of samples may be necessary in order to obtain significance.
Animals↗
Determination of olsalazine sodium in pharmaceuticals by differential pulse voltammetry.
The electrochemical oxidation of olsalazine sodium was investigated by cyclic, linear sweep, differential pulse and square wave voltammetry using glassy carbon disc electrode in different buffer systems. Best results were obtained for the determination of olsalazine using the differential pulse voltammetric technique in phosphate buffer at pH 7.0. The electroactive species exhibits a diffusion-controlled voltammetric wave and its differential pulse peak current shows a linear dependence on olsalazine concentration in the range between 2 x 10(-6) M and 2 x 10(-4) M. This relationship has been applied to the determination of olsalazine in commercial capsule dosage forms. The recovery study shows good accuracy and precision for the assay developed. A UV spectrophotometric assay is also reported for comparison.
Aminosalicylic Acids↗
Newly developed TaqMan assay to detect West Nile viruses in a wide range of viral strains.
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Humans↗
Approaches to the development of assays for interaction of tobacco-specific nitrosamines with haemoglobin and DNA.
The tobacco-specific, nicotine-derived nitrosamines 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are among the most important carcinogens in tobacco and tobacco smoke. Treatment of Fischer 344 rats with these carcinogens resulted in alkylation of haemoglobin and DNA by the 4-(3-pyridyl)-4-oxobutyl group formed during their metabolism. This alkyl group can be detached from globin or DNA under mild hydrolytic conditions as 4-hydroxy-1-(3-pyridyl)-1-butanone, which appears to be a potentially useful dosimeter for human exposure to, and activation of, tobacco-specific nitrosamines.
Animals↗
Studies on serum lipase against different triglycerides in some clinical conditions. 2. Purification of triolein and tristearin and development of assay method for trioleinase and tristearinase.
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Enzymes↗
The immunoreactivity profile of different HCV genotypes on immunoblot assay and its implications in the development of diagnostic assays.
The immunoreactivity profiles of plasma samples obtained from patients infected with different hepatitis C virus (HCV) genotypes were studied using immunoblot assay containing multiple HCV antigens. The immunoblot assay was found to be positive in 81.5% of 195 blood donors who had anti-HCV antibodies as detected by second generation enzyme immunoassays. The samples reacted preferentially with the viral core, NS3-1 and NS5 antigens, and these reactivities were not influenced by HCV genotype. However, the reactivities with NS3-2 and NS4 antigens varied depending on HCV genotypes. The samples from patients infected with HCV genotype 1 reacted well with NS3-2 and NS4 antigens whereas those with other genotypes did not. In addition, samples with the unclassified HCV genotype reacted poorly with all antigens, except NS3-1. This study demonstrates the importance of the core, NS3-1 and NS5 antigens in the detection of antibodies against HCV, especially in areas where more than one genotypes of HCV are present. It also demonstrates that there is a need for further improvement of the currently used assays as new HCV genotypes are recently discovered.
Genotype↗
Plasma progesterone in cattle. I. Development and validity of the assay.
The development of a practical competitive protein-binding assay for plasma progesterone in cattle is described. With an intra-assay coefficient of variation of 5,46% and an interassay coefficient of variation of 14,25%, the method is sufficiently accurate and sensitive for practical purposes, and for use in routines and surveys. The statistical level of sensitivity was found to be in the region of 0,25 ng/ml based on the confidence limits of zero dose and 0,25 ng/ml, with the practical sensitivity level at 0,50 ng/ml. Method and reagent blanks were found to be negligible. The specificity of the assay is based entirely on the partial specificity of the petroleum ether used for the extraction of progesterone (87,5% extraction, n = 141).
Animals↗
Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research.
OBJECTIVE: We developed assays for measurement of urinary betaLH and betaFSH under collection and storage conditions typical of non-clinical research settings. DESIGN AND METHODS: IEMAs for free betaLH and total betaFSH were validated by standard methods. Stability of urinary betaLH and betaFSH was tested across freeze-thaws and stored long term at 4 degrees C or -20 degrees C, or short term at room temperature, and with heating to dissociate the subunits. RESULTS: The IEMAs exhibited acceptable parallelism, specificity, recovery (averaging 100% for betaLH, 97% for betaFSH), imprecision (maximum within-run and between run CVs, respectively, 4.8% and 25.7% for betaLH, 5.6% and 17.0% for betaFSH), and minimum detectable dose (2.5 pmol/L for betaLH, 6.8 pmol/L for betaFSH). Urine and serum measures were highly correlated (r=0.95 for LH, 0.86 for FSH). There was no consistent decline with any storage type. Dissociation of subunits by heating was needed for betaLH, but not betaFSH. CONCLUSION: These IEMAs measure free betaLH and total betaFSH, overcoming inter-individual variability in, and collection and storage effects on, subunit dissociation, without the need for urine preservatives.
Adult↗
Analysis of zebrafish development using explant culture assays.
Two fundamental questions of developmental biology are when cells become committed to a certain lineage and what cell interactions are involved in establishing this commitment. These questions can be answered using explant or transplant assays. We have developed explant assays to study zebrafish development. These assays involve isolating by microdissection small regions of the embryo at specific times during development, and determining their fate after culture in isolation (lineage commitment assays) or after exposure to a putative inducing tissue (induction assays). In our laboratory, we have used these assays to address questions of neural development, including those of commitment to anterior and posterior neural lineages, and the signals involved in making these decisions. This chapter contains detailed guidelines for designing explant assays. These include suggestions for the isolation and successful culture of explants, and descriptions of the methods used to assay the final fate of explants after culture. Step-by-step protocols are given for the isolation of specific explants that can be used in specification and induction assays. The application of this technique is illustrated with descriptions of experiments. Explant assays will continue to generate key information concerning the establishment of lineage commitment of many embryonic tissues and will provide extremely valuable for analysis of new genes identified molecularly and in mutant screens.
Animals↗
Simple strategies for reducing sample loads in in vitro metabolic stability high-throughput screening experiments: a comparison between traditional, two-time-point and pooled sample analyses.
Higher-throughput ADME programs in early drug discovery are becoming common throughout the pharmaceutical industry as companies strive to reduce their compound attrition in later-stage development. Many of the ADME assays developed into higher-throughput formats rely on LC/MS analyses. Since the biological aspects of the assay are amenable to parallel processes using dense plate formats, the number of samples generated from these assays produce a large analysis load for serial LC/MS. Presented in this report are two novel strategies, including a sample pooling method and a two time-point method, that could be used in drug discovery to reduce the number of samples generated during multiple time-point in-vitro ADME assays. One hundred and sixty-three compounds were subjected to human microsomal incubations with full time-point method samples taken at t = 0, 5, 15, 30, and 45 min. The ER data correlation (R(2)) between the full time-point method and the pooling method and two time-point methods were 0.98 and 0.97, respectively. Both methods have the potential to: 1. produce data of similar quality to traditional high throughput ADME assays, 2. be easily implemented, 3. shorten analytical run times, and 4. be reproducible and robust.
Algorithms↗
'One bead two compound libraries' for detecting chemical and biochemical conversions.
When combinatorial chemistry was introduced 13 years ago, the expectations were high for the delivery of results, particularly in the pharmaceutical industry. However, combinatorial chemistry was implemented independently of the application for which the products were going to be used. Resins developed only for efficient solid-phase synthesis were used and products were employed in existing assays developed for traditional solution studies. There was almost no assay or technology development and the use of real combinatorial methods soon had to give way to high-throughput synthesis and traditional screening. However, during recent years more sophisticated resins and assay techniques have been developed that may result in a second and more successful implementation of real integrated combinatorial chemistry. The first in this line of new developments is the 'one bead two compound' assay, in which the resin bead in addition to a combinatorial library member contains a reporter compound that can act as a beacon to monitor the activity of the library member. This powerful concept can be generally applied in all fields of combinatorial chemistry including drug, catalysts and material development.
Catalysis↗
High-throughput single nucleotide polymorphism genotyping by fluorescent 5' exonuclease assay.
Single nucleotide polymorphisms (SNPs) are the class of DNA variants that are most common in the genome. Technologies are needed that allow relatively high-throughput, high-quality genotyping of SNPs, with rapid assay development for individual SNPs of interest. We have implemented an accurate and high-throughput SNP genotyping system using a commercially available fluorescent 5' exonuclease assay. Optimization of the assay system for low-volume reactions and low-probe concentrations has reduced assay costs by 75%. Using a simple assay optimization process, we successfully developed genotyping assays for 92% of assays attempted (309 out of 335) and have generated over 200,000 genotypes.
Alleles↗
Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay.
The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.
ATP-Binding Cassette Transporters↗
[A method of fluorescence immunoassay with temporal resolution and the use of europium label and polymeric complexon].
Recently developed solid-phase immunofluorescent assay with temporal distinction involved a newly produced procedure for binding of of the boron group label with antibodies via modified polymer, which is able to bind with antibodies across the avidin bridge. The assay developed is more simple than the widely used procedures, it exhibits high sensitivity being superior as compared with a corresponding immunoenzyme assay by a decimal order.
Animals↗