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PubMed1976

Development of an assay for aryl hydrocarbon (benzo(a)pyrene) hydroxylase in human peripheral blood monocytes.

An assay has been developed and measurements of aryl hydrocarbon [benzo(a)pyrene] hydroxylase have been made in peripheral blood monocytes from a human population. Treatment with benz(a)anthracene in cell culture increased aryl hydrocarbon hydroxylase activity from 6.5 to 37-fold in monocytes from each of 25 apparently healthy donors. A weak correlation (r = 0.38) was observed between the induction ratios obtained with monocytes and lymphocytes from the same donors. Reproducibilities of the monocyte and lymphocyte assays were comparable. In monocytes, the measurement of basal and induced aryl hydrocarbon hydroxylases activity does not require pretreatment with mitogens as is the case with lymphocytes. Monocytes also exhibit a much wider range of induction ratios than do lymphocytes.

Adult↗

PubMed1986

Development of an assay system for the detection and classification of methotrexate resistance in fresh human leukemic cells.

An assay system was developed for the detection and classification of methotrexate resistance in fresh human leukemic cells. Mechanisms of resistance to be identified were: overexpression of dihydrofolate reductase, decreased cellular uptake of methotrexate, decreased affinity of dihydrofolate reductase for methotrexate, decreased polyglutamylation of methotrexate, and low thymidylate synthase activity. The initial screening procedure utilizes 3H release after addition of [5-3H]-2'-deoxyuridine as a measure of intracellular activity of thymidylate synthase and of DNA synthesis; 3H release is assayed after 3-h incubations with methotrexate, trimetrexate, or gamma-fluoromethotrexate and after 4-h incubations with these agents followed by a 6-h incubation in drug free medium. The pattern of DNA synthesis inhibition and recovery under these two sets of conditions establishes the presence or absence of methotrexate resistance and allows a tentative classification of the resistance mechanism involved. In combination with determinations of dihydrofolate reductase activity, methotrexate titration studies, and the determination of intracellular drug accumulations in vitro, the system is readily able to classify CCRF-CEM human leukemia cell lines possessing well defined mechanisms of resistance. The findings in seven leukemia patients are also reported. Applying tentative reference values, four patients showed biochemical evidence of methotrexate resistance: two patients had only a transport defect, one patient had evidence of both a transport defect and low thymidylate synthase activity, and one patient appeared to have decreased methotrexate polyglutamylation. Application of the assay system in larger numbers of patients is feasible and is required to establish adequate reference values for the evaluation of biochemical-clinical correlates.

Adolescent↗

PubMed2005

Development of an assay for the quantification of type I collagen synthesis in the guinea pig.

There is a need for a reliable assay for the quantification of collagen type I synthesis in the guinea pig, an important model for many connective tissue diseases. Procollagen type I C-terminal propeptide (PICP) is the established marker of type I collagen synthesis but, to date, no assay has been developed to measure PICP in guinea pig tissue extracts. A monoclonal antibody, known to cross-react with intact guinea pig procollagen type I (anti-PICP), was tested for its ability to bind soluble guinea pig PICP in crude skin extracts using a biosensor. Anti-PICP was immobilised to the surface of a sensor chip and antibody-antigen binding was detected using the phenomenon of surface plasmon resonance (SPR). The binding component in the SPR-immunoassay was identified as PICP by purification and N-terminal sequencing. Guinea pig PICP was purified from skin by gel filtration, ion exchange chromatography and lectin affinity chromatography. Purified PICP was then biotinylated and used with anti-PICP to develop a competition ELISA that was able to selectively and sensitively measure PICP in extracts of guinea pig connective tissue.

Animals↗

PubMed1987

Critical considerations in the development of an assay for sulfidopeptide leukotrienes in plasma.

A sensitive and specific assay has been developed for measurement of total sulfidopeptide leukotrienes (LT) in plasma. LTC4 and LTD4 in plasma are converted to LTE4 which is then extracted by C18 Sep-Pak binding and elution. Total LTE4 is resolved by reverse phase high performance liquid chromatography (RP-HPLC) and quantitated by radioimmunoassay (RIA). A [3H]LTE4 internal standard is added to the starting plasma sample to allow overall recovery to be calculated and to define the fractions from RP-HPLC to be assayed for LTE4-like immunoreactivity. The correlation between the measured increase in LTE4 concentration after addition of incremental amounts of LTC4 and LTE4 to plasma was 0.989 and 0.978, respectively, with slopes of 1.05 and 1.11. Addition of 51 pg/ml LTE4 to 5 ml plasma was detectable; the measured increase was 48 +/- 12 pg/ml (mean +/- SE, n = 7). The intra-assay coefficient of variation for 341 pg/ml of added LTC4 was 3.2% (n = 6). Sulfidopeptide leukotrienes could not be detected in blood samples taken from 12 normal volunteers in whom the theoretical detection limit, calculated from the sensitivity of the RIA, the overall recovery of LTE4, and the volume of plasma extracted, was 83 +/- 4 pg LTE4/ml plasma (0.19 +/- 0.01 pmol sulfidopeptide leukotriene/ml plasma; mean +/- SE).

Adult↗

PubMed2005

Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales.

OBJECTIVES: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in Helicobacter pylori isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. METHODS: Tetracycline susceptibility was determined by disc diffusion. The MIC of isolates with reduced susceptibility was determined by Etest and agar dilution methods. The 16S rDNA of these isolates was sequenced and resistance-associated mutations identified. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture and gastric biopsies. RESULTS: From 1006 isolates of H. pylori examined, 18 showed reduced susceptibility to tetracycline. Of these, three were resistant (>or=4 mg/L). Mutations in 16S rDNA were detected in 10 of the reduced susceptibility isolates: one double base mutation of A926T/A928C, one A926C, one A928C; and seven A926G. The first two polymorphisms were novel and had not been reported from clinical isolates previously. The LightCycler assay identified each of the 10 isolates with 16S rDNA mutations, but did not detect polymorphisms in 100 tetracycline-susceptible H. pylori isolates. The assay correctly determined the tetracycline susceptibility of H. pylori in 20 gastric biopsy samples. CONCLUSIONS: Mutations in 16S rDNA were detected in H. pylori isolated in England and Wales with reduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.

DNA, Ribosomal↗

PubMed2002

Development of an assay for beta-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein.

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, beta-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and beta-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. beta-Lactamase catalyzes the hydrolysis of the beta-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the beta-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of beta-lactamase in a format that should be amenable to high-throughput screening.

Aequorin↗

PubMed1983

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay.

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.

Animals↗

PubMed2001

A strategy for quantitative bioanalysis of non-polar neutral compounds by liquid chromatography/electrospray ionization tandem mass spectrometry: determination of TS-962, a novel acyl-CoA:cholesterol acyltransferase inhibitor, in rabbit aorta and liver tissues.

A strategy for the sensitive and reliable quantitative determination of non-polar neutral compounds in biological matrices by liquid chromatography/electrospray ionization tandem mass spectrometry is described in the context of assay development for TS-962, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rabbit aorta and liver tissues. The electrospray ionization (ESI) mass spectrum of this compound with a mobile phase of water/acetonitrile did not give abundant [M + H]+ ions, but did give alkali metal cation adducts such as [M + Na]+, [M + CH3CN + Na]+ and [M + K]+ ions. The cationized species are acknowledged as unsuitable precursor ions for selected reaction monitoring (SRM) for various reasons, such as difficulty in obtaining characteristic product ions in low-energy collision-induced dissociation, and irreproducibility of the adduct-ion intensities. To overcome this problem, a solution of 3.4 mM trifluoroacetic acid in 2-propanol was added to the mobile phase as a postcolumn additive, resulting in a decrease of the undesirable adduct formation and significant enhancement of [M + H]+ ion intensity. An attempt was then made to prevent the matrix effect by employing a column-switching system, which allowed direct injection of a large volume of 2-propanolic tissue homogenate (950 microL) followed by sufficient clean-up, separation, and ESI-SRM on-line. This enabled development of a sensitive and reliable assay method for TS-962 in rabbit aorta and liver tissues in the concentration range of 5-500 ng/g wet tissue using a 25-mg aliquot of tissue sample. Application of this method to the determination of aortic TS-962 levels at 24 h after repeated oral administration of this compound (3 mg/kg) once a day for 12 weeks to 1% cholesterol-fed rabbits is also presented. Results showed that TS-962 is well distributed to both the thoracic and abdominal aorta tissues, at levels higher than the 50% inhibitory concentration value of this compound for microsomal ACAT activity from rabbit aorta.

Acetamides↗

PubMed2004

Development and validation of an HPLC method for AG331 bulk drug substance and lyophilized powder for injection.

AG331 is a water soluble glucuronate salt of a novel antitumor compound synthesized by protein structure based drug design. A lyophilized powder for injection was developed for clinical studies. During HPLC assay development, AG331 showed an inherent tailing problem due to an amino group in the structure. An optimized reverse-phase gradient HPLC method was developed to minimize the tailing and separate AG331 from its synthetic intermediates (I-1, I-2, I-3, I-4, I-5, I-6, I-8), other impurities and degradation compounds. The method was shown to be linear, precise, accurate, rugged and stability-indicating.

Chromatography, High Pressure Liquid↗

PubMed2001

Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene.

BACKGROUND: In different populations of the world, more than 150 genetic alterations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in different ethnic communities, none of the assays developed for screening mutations/deletions in a population defined can be adapted to study the possible genetic defects. The present study was designed to develop a new, multiplex PCR-based, molecular biological method to screen the whole coding region of the LDL receptor gene. METHODS: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83-386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by agarose gel electrophoresis/ethidium bromide staining. RESULTS: A rapid, effective test enabling the screening of DNA alterations in the entire LDL receptor gene was developed. Using this simple multiplex PCR assay, deletions affecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. CONCLUSIONS: The simplicity, specificity and versatility of the assay make it suitable system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening of LDL-RG deletions in populations or groups at high risk for cardiovascular diseases.

Electrophoresis, Agar Gel↗

PubMed1997

Functional assessment of bovine monocytes isolated from peripheral blood.

Bovine monocytes were isolated from the peripheral blood of cattle by adherence of peripheral blood mononuclear cells to plastic. Three in vitro methods were modified to evaluate bovine monocyte function. These methods were: (1) ingestion of 125I-iododeoxyuridine labeled Staphylococcus aureus; (2) antibody-dependent cell-mediated cytotoxicity; (3) luminol-dependent and native chemiluminescence. Description of monocyte isolation and assay development is discussed in the text. Assays to evaluate monocyte function are useful for assessing immune status of the animal.

Animals↗

PubMed1996

Genetic analysis of the human cytochrome P450 CYP2C9 locus.

Cytochrome P450 CYP2C9 metabolizes a wide variety of clinically important drugs, including phenytoin, tolbutamide, warfarin and a large number of non-steroidal anti-inflammatory drugs. Previous studies have shown that even relatively conservative changes in the amino acid composition of this enzyme can affect both its activity and substrate specificity. To date six different human CYP2C9 cDNA sequences, as well as the highly homologous CYP2C10 sequence have been reported suggesting that the CYP2C9 gene is polymorphic. Only nine single base substitutions in the coding region of CYP2C9 account for the differences seen between the CYP2C9 proteins. In this report we have developed polymerase chain reaction (PCR)-based assays to distinguish all seven sequences, and have determined their allele frequencies in the Caucasian population. Of the seven sequences studied in one hundred individuals only three appeared to be CYP2C9 alleles. These alleles termed CYP2C9*1, CYP2C9*2 and CYP2C9*3 had allele frequencies of 0.79, 0.125 and 0.085 respectively. The CYP2C10 gene could not be found in any of the samples studied. The assays developed here will allow the prediction of CYP2C9 phenotype, thus identifying those individuals who may exhibit different drug pharmacokinetics for CYP2C9 substrates.

Alleles↗

PubMed1994

Heterogeneous expression and polymorphic genotype of glutathione S-transferases in human lung.

BACKGROUND: Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics by conjugation with glutathione. One of the mu class genes of this superfamily of enzymes, GSTM1, is polymorphic because of a partial gene deletion. This results in a failure to express GSTM1 in approximately 50% of individuals. Several studies have linked GSTM1 null status to an increased risk of lung carcinoma. This study investigated the expression and distribution of GST isoenzymes in human lung, and developed a polymerase chain reaction (PCR) assay which would allow genotyping of archival, paraffin embedded lung tissue. METHODS: Distribution was examined using a panel of polyclonal anti-GST antibodies for immunohistochemistry in normal tissue of 21 tumour-bearing lungs. DNA for PCR was extracted from paraffin blocks and a control group of 350 blood lysates. As a positive control each assay amplified part of GSTM4, a mu class gene which is not polymorphic but which shows strong sequence homology to GSTM1. The presence of GST in bronchoalveolar lavage fluid was sought by Western analysis. RESULTS: Proximal airways contained pi class GST, alpha class GST, and mu class GST with expression concentrated in the brush border. In distal airspaces no alpha GST was expressed but pi GST and mu GST were present in alveolar cells and also alveolar macrophages. Pi class GST was present in bronchoalveolar lavage fluid. The PCR assay enabled genotypic determination using DNA extracted from archival material. Of the control group 56% were null at the GSTM1 locus. CONCLUSIONS: The distribution of GST isoenzymes in the lung is heterogeneous with an apparent decrease in GST in distal lung. Since GSTM1 status has already been associated with susceptibility to disease, the PCR assay developed will allow further studies of the relation between genotype and structural disorders in the lung using archival pathological material.

Base Sequence↗

PubMedDate not supplied

Comparison of antiproliferative effects of experimental and established antipsoriatic drugs on human keratinocytes, using a simple 96-well-plate assay.

Pharmacological treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of two or more of these actions. Potentially new drugs for treatment of psoriasis, which act on proliferation, can be identified by screening large compound libraries in a cell proliferation model that allows for characterization of drug effects on in vitro growth of normal human keratinocytes. High-throughput programs based on biological testing of diverse collections of compounds can rapidly identify leads for potential drug candidates in the treatment of psoriasis. In this study, we describe nonradioactive measurement of keratinocyte proliferation in the exponential growth phase in a 96-well format, using a sensitive deoxyribonucleic acid-binding dye to analyze drugs that are pharmacologically active in growth inhibition. Release of lactate dehydrogenase was used to exclude cytotoxic effects. We examined a number of compounds in a test range of 10(-7) to 10(-5) M, including known antipsoriatic drugs, and experimental drugs that are potentially useful in the treatment of psoriasis. We found strong concentration-dependent growth inhibition by dithranol, an antipsoriatic compound that is presumed to target the epidermal compartment. Methotrexate, cyclosporin A, and all-trans retinoic acid did not significantly affect proliferation at therapeutically relevant concentrations. The p38 mitogen-activated protein kinase inhibitor, SB220025, and curcumin, a natural phytochemical, inhibited keratinocyte proliferation at 10(-5) M. We conclude that this assay, in combination with the previously developed assays for psoriatic differentiation, provides a useful tool for identification of antipsoriatic drugs.

Anthralin↗

PubMed2001

Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes.

PURPOSE: Unexpected reverse transcription-PCR detection of cytokeratin 20 (CK20) in samples from healthy individuals and cancer types not expected to express CK20 has cast uncertainty on the role of CK20 as a specific marker of disseminated colorectal cells. We aimed to clarify the specificity of CK20 by examining its expression profile by real-time reverse transcription-PCR. EXPERIMENTAL DESIGN: A quantitative real-time PCR assay on the LightCycler instrument was developed and used to examine CK20 expression in tumors and lymph nodes from subjects with colorectal and breast carcinoma, head and neck and vulval squamous cell carcinoma, and melanoma. To select a method for reproducible quantification, four approaches were evaluated. RESULTS: The developed assay allowed rapid, convenient-to-use, specific, sensitive, and reproducible CK20 quantification amenable to large-scale analysis. For quantity calculation, an efficiency-adjusted relative ratio method was selected that controls for RNA loading and integrity as well as inefficient PCR reactions and provides a platform for standardization across laboratories. Using this assay, we detected CK20 in 41 of 89 (46%) lymph nodes from noncolorectal cancer types. There was a strong association between CK20 detection and lymph node metastasis determined by histology (P < 0.0001). Quantitatively, CK20 expression levels in colorectal cancer lymph nodes significantly exceeded the levels obtained in lymph nodes of extracolonic carcinomas (P < 0.05). Mean CK20 levels in lymph nodes and tumors from subjects with colorectal and breast cancers were similar in a tumor-type specific fashion. CONCLUSIONS: These results characterize low-level, epithelial cell-specific CK20 expression in infiltrated lymph nodes from subjects with noncolorectal cancer types and demonstrate the potential advantages of detecting circulating epithelial cells by quantitative PCR.

Biomarkers, Tumor↗

PubMed1994

Quantitative diagnosis of Schistosoma infections by measurement of circulating antigens in serum and urine.

Recent research on the detection of the schistosome circulating antigens, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), has greatly expanded the scope of applications. In the present paper aspects of assay development, in particular of highly sensitive assays and of field-applicable assays, are discussed. The progress in the use of CAA- and CCA-detection for diagnosis, follow-up of chemotherapy and sero-epidemiological studies is evaluated.

Animals↗

PubMed1994

Development of an assay method for purine catabolic enzymes in the mouse and its adaptation for use on an autoanalyzer.

An assay method has been developed for the purine catabolic enzymes adenosylhomocysteinase, adenosine deaminase (ADA), purine-nucleoside phosphorylase (PNP), and urate oxidase in mice. The assay links H2O2 produced during purine catabolism to the production of a dye complex. The assay method has been developed for ADA and PNP in erythrocytes and for all four enzymes in liver. The assay is cheap, sensitive, and easy to perform. The dye complex absorbs in the visible range, negating the need for an expensive ultraviolet spectrophotometer and allowing the use of an autoanalyzer.

Adenosine Deaminase↗

PubMed2002

Health care industries' perspective of viral load assays: the VERSANT HIV-1 RNA 3.0 assay.

This article will compare the VERSANT HIV-1 RNA 3.0 (bDNA 3.0) assay with other HIV-1 viral load assays, particularly the AMPLICOR HIV-1 MONITOR (Amplicor 1.5), the industry standard. The discussion will cover the history of viral load assay development and challenges to the field. It will finish with a description of the evolving markets for viral load assays in the developing world and the impact of variations in the different assays on their ability to reach those markets and patient populations.

Automation↗