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At least 793 records · Page 44Linked to original sources

PubMed1994

Application of a transformed cell line constitutively expressing HSV-1 polypeptides for the detection of HSV antibodies in human sera by an enzyme immunoassay.

We have previously reported the construction of a cell line BA4, constitutively producing the glycoproteins gD, gG, and alpha 4, the major regulatory protein of HSV-1. These cells have been selected in stepwise increasing concentrations of methotrexate and shown to produce much higher amounts of gD than non-selected cells. Extracts of the selected cells were used in an enzyme linked immunosorbent assay to detect HSV antibodies in human sera obtained from Greek blood donors. We report here that (i) the assay developed is able to distinguish HSV antibody positive from negative human sera and (ii) that its application in an epidemiological survey showed that the incidence of HSV infection in the general population in Greece is 90.4%.

Animals↗

PubMed2004

Development of a multiplex PCR for detection of avian adenovirus, avian reovirus, infectious bursal disease virus, and chicken anemia virus.

A multiplex polymerase chain reaction (mPCR) was developed and optimized for the simultaneous detection and differentiation of avian reovirus (ARV), avian adenovirus group I (AAV-I), infectious bursal disease virus (IBDV), and chicken anemia virus (CAV). Four sets of specific oligonucleotide primers were used in this test for ARV, AAV-I, IBDV, and CAV. The mPCR DNA products were visualized by gel electrophoresis and consisted of fragments of 365 bp for IBDV, 421 bp for AAV-I, 532 bp for ARV, and 676 bp for CAV. The mPCR assay developed in this study was found to be sensitive and specific. Detection of PCR-amplified DNA products was 100 pg for both CAV and IBDV, and 10pg for both ARV and AAV-I and this mPCR did not amplify nucleic acids from the other avian pathogens tested. The mPCR demonstrated similar sensitivity in tests using experimental fecal cloacal swab specimens that were spiked with ARV, AAV-1, IBDV, and CAV, and taken from specific pathogen free (SPF) chickens. This mPCR detected and differentiated various combinations of RNA/DNA templates from ARV, AAV-I, CAV, and IBDV without reduction of amplification from feces.

Animals↗

PubMed2002

Immunofluorescence detection of Escherichia coli in seawater: a comparison of various commercial antisera.

Through a microscopical method, relying on the interaction between fluorescent antibodies and target antigen, it is possible to detect and enumerate Escherichia coli in seawaters. Various commercial monoclonal and polyclonal antisera have been tested in an indirect immunofluorescence (IIF) assay developed for microbiological monitoring of coastal waters. Prior to use, they have been titrated and screened for cross-reactions with a collection of clinical and environmental isolates. A comparison among counts obtained on field samples showed higher performance for microscopical than for plate methods, due to the ability of all antisera to label target cells specifically, regardless of their viability. Because of their different specificities, polyclonal antisera yielded better quantitative results than monoclonal antisera. The study further suggested the usefulness of the immunofluorescence assay as a rapid alternative analytical tool for the specific detection of bacterial pathogens in aquatic environments.

Colony Count, Microbial↗

PubMed1993

Comparison of in vitro and in vivo biological activity of mycotoxins.

In vitro assays developed to screen the cytotoxic activity of chemicals in murine (NIH/3T3) and bovine (BE 12-6) embryonic cells were used to determine the concentrations (microgram/ml) of mycotoxins which caused 50% lethality (LC50). Embryonic cells were seeded in 96 well plates, cultured for 72 hr with dilutions of each individual and combinations of mycotoxins, and stained and counted. Verrucarin A and roridin A had the strongest cytotoxic activity, and ergotamine tartrate was least toxic. Furthermore, results correlated with published values of in vivo activity, indicating this assay can be used for acute toxicity screening of compounds.

3T3 Cells↗

PubMed1998

Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma.

Substantial evidence indicates that triglyceride-rich lipoprotein remnants are atherogenic. Additional research has, however, been limited by available methods for separation and quantification of remnants. We have evaluated an immunoseparation assay developed to measure cholesterol in remnant-like particles (RLP-C). This method uses monoclonal antibodies to human apolipoproteins B-100 and A-I to remove most of the apolipoprotein B-100-containing lipoproteins (namely LDL and nascent VLDL) and apolipoprotein A-I-containing lipoproteins (namely chylomicrons and HDL), leaving behind a fraction of triglyceride-rich lipoproteins, including chylomicron and VLDL remnants, both of which are enriched in apolipoprotein E. Cholesterol in the unbound fraction is measured with a sensitive enzymatic assay. The RLP-C concentration was highly correlated with total triglyceride-rich lipoproteins (sum of VLDL-cholesterol and IDL-cholesterol) separated by ultracentrifugation and by polyacrylamide gel electrophoresis (r = 0.86 and 0.76, respectively). The within-run and run-to-run imprecision (CV) of the assay was approximately 6% and 10%, respectively. The assay was not affected by hemoglobin up to 5000 mg/L (500 mg/dL), bilirubin up to 342 mmol/L (20 mg/dL), glucose up to 67 mmol/L (1200 mg/dL), or ascorbic acid up to 170 mmol/L (3.0 mg/dL). In 726 subjects (men, n = 364; women, n = 362) in the US, the 75th percentiles of RLP-C concentration were 0.17 mmol/L (6.6 mg/dL) and 0.23 mmol/L (8.8 mg/dL) in sera obtained after overnight fasting or randomly, respectively. A group of 151 patients from nine US centers and one Canadian center with coronary artery atherosclerosis established by angiography had higher median RLP-C concentrations than 302 gender- and age-matched controls (P <0.05). We conclude that the RLP-C assay compares favorably to ultracentrifugation and electrophoresis and provides a convenient and economical approach to measure triglyceride-rich lipoprotein remnants in routine clinical laboratories.

Adolescent↗

PubMed1997

Prediction of left ventricular mass changes after renal transplantation by polymorphism of the angiotensin-converting-enzyme gene.

Cardiac complications are the main cause of death in renal transplant patients and left ventricular hypertrophy (LVH) may play a determinant role. An association between the insertion-deletion polymorphism of the angiotensin-converting enzyme (ACE) gene and LVH has been reported in adults. However, little is known about the genetic influence on left ventricular mass changes after renal transplantation, where unique environmental factors, such as cyclosporine A (CsA) and prednisone treatment concur. In fact, CsA treatment has recently been associated with the development of LVH. We prospectively determined the changes on cardiac structure and function, assessed by echocardiographic criteria, in 38 consecutive nondiabetic adults who received a cadaveric renal allograft. They were treated with cyclosporine and prednisone and maintained a good renal function during the follow-up. Echocardiographic studies (M-mode, 2-B and color flow Doppler) were performed without previous knowledge of the genetic typing, at the time of transplantation, and 6 and 12 months later. ACE alleles were typed using a PCR-based assay developed to ascertain the presence of an insertion (I)-deletion (D) polymorphism in intron 16 of the ACE gene. Patients with the so-called "unfavorable" DD genotype (N = 16) were compared with the ID or II genotypes (N = 22). The baseline left ventricular mass index was similar in patients with or without the unfavorable DD genotype (X +/- SE; 166.6 +/- 10.4 vs. 181.3 +/- 9 g/m2, respectively) and a similar proportion fulfilled the criteria of LVH (88% vs. 82%, respectively). The mean percent increase of the left ventricular mass index 12 months after renal transplantation was significantly higher in patients with the DD genotype compared to those with other genotypes (21.3 +/- 7.9 vs. -0.08 +/- 4.9%, respectively; P < 0.05). As a result, 94% of DD patients showed LVH at the end of the follow-up, while 68% of the ID or II patients had LVH (P < 0.05). In addition, the left ventricular ejection fraction significantly increased only in ID or II patients 12 months after transplantation with respect to baseline (ID/II patients, 70.4 +/- 1.5 vs. 63.7 +/- 1.8%; P < 0.05; DD patients, 68.3 +/- 2.1 vs. 63.3 +/- 2.9%). The deleterious effect of the DD genotype was independent of blood pressure, biochemical parameters, weight gain, and cumulative steroids dosages or cyclosporine levels. In conclusion, genetic factors determine the changes on cardiac structure and function after renal transplantation. The presence of the DD genotype of the ACE gene is a marker associated with an elevated risk of LVH in this population.

Adult↗

PubMed1998

Kinetic analysis and tissue distribution of human D-glycerate dehydrogenase/glyoxylate reductase and its relevance to the diagnosis of primary hyperoxaluria type 2.

The enzyme D-glycerate dehydrogenase (D-GDH; EC 1.1.1.29), which is also believed to have glyoxylate reductase (GR; EC 1.1.1.26/79) activity, plays a role in serine catabolism and glyoxylate metabolism and deficiency of this enzyme is believed to be the cause of primary hyperoxaluria type 2 (PH2). The pH optima and kinetic parameters of D-GDH and GR in human liver have been determined and assays developed for their measurement. Maximal activities were observed at pH 6.0, 8.0 and 7.6 for the D-GDH forward, D-GDH reverse and GR reactions, respectively. The apparent Km values for the substrates in these reactions were as follows: D-GDH forward reaction, 0.5 mmol/L hydroxypyruvate and 0.08 mmol/L NADPH; D-GDH reverse reaction, 20 mmol/L D-glycerate and 0.03 mmol/L NADP and for the GR reaction 1.25 mmol/L glyoxylate and 0.33 mmol/L NADPH. The forward D-GDH and GR assays were adopted for routine use, the low activity of the reverse D-GDH reaction being of little use for routine analyses. D-GDH and GR activity in 13 normal livers ranged from 350-940 nmol per min per mg protein (median 547) and 129-209 nmol per min per mg protein (median 145), respectively. D-GDH activity in kidney, lymphocytes and fibroblasts fell within the range of values seen in the liver but GR activity was approximately 30% in the kidney and barely detectable in lymphocytes and fibroblasts. Analysis of liver and lymphocyte samples from patients with PH2 showed that GR activity was either very low or undetectable while D-GDH activity was reduced in liver but within the normal range in lymphocytes. These results suggest that there is more than one enzyme with D-GDH activity in human tissues but only one of these has significant GR activity. We conclude that a definitive diagnosis of PH2 requires measurement of GR and D-GDH in a liver biopsy.

Alcohol Oxidoreductases↗

PubMed1985

Immune complexes containing factor V in a patient with an acquired neutralizing antibody.

An 82-year-old woman presented with extensive hematomas and melena associated with markedly decreased plasma factor V coagulant activity (FV:C). Using a competitive enzyme-linked immunosorbent assay developed in our laboratory, we made serial measurements of factor V antigen (FV:Ag) in plasma and found it to be normal or elevated. The patient's plasma was demonstrated to contain an IgG antibody that could neutralize FV:C in normal plasma. The antibody was of restricted heterogeneity (IgG1, IgG2,kappa). Circulating immune complexes containing antibody to factor V and FV:Ag were demonstrated directly in the plasma by immunoelectrophoresis with polyclonal monospecific antibody and with a monoclonal antibody using an enzyme-linked immunosorbent assay. Presence of neutralizing antibody could be demonstrated in vitro even at times when FV:C was within normal limits by heat inactivation of FV:C. Treatment with plasma and platelet transfusions as well as plasmapheresis induced definite but transient elevation of FV:C. Steroid therapy lowered the neutralizing antibody concentration and produced a rapid and persistent elevation of FV:C during two separate hospitalizations. This report describes a patient in whom levels of FV:Ag have been serially measured, and the presence of circulating immune complexes consisting of factor V and a neutralizing antibody have been directly demonstrated.

Aged↗

PubMed2000

Novel assay for Roberts syndrome assigns variable phenotypes to one complementation group.

Roberts syndrome (RS) is a rare autosomal recessive disorder characterized by heterogeneous clinical features, the most notable being tetraphocomelia, cleft lip, and cleft palate. Cells derived from most RS patients exhibit abnormal cytogenetic and cellular phenotypes that include the premature separation of para- and pericentromeric heterochromatin visible on C-banded metaphase chromosomes, a phenomenon referred to as heterochromatic splaying. Previously, it was shown that these abnormal phenotypes can be complemented following somatic cell hybridization between RS cells and control cells. In the current study, a permanent cell line was established from a new RS patient with a more severe phenotype than represented by previously established cells in culture. With a newly developed assay designed to facilitate rapid evaluation of in vitro complementation, we assigned this new patient to the same genetic complementation group defined by other, less severely affected patients. The results demonstrate that a single complementation group defines RS patients with heterochromatic splaying regardless of clinical severity.

Abnormalities, Multiple↗

PubMed1997

Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay.

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.

Amphotericin B↗

PubMed2003

An antisense construct reduces N-methyl-D-aspartate receptor 2A expression and receptor-mediated excitotoxicity as determined by a novel flow cytometric approach.

The N-methyl-D-aspartate receptor (NMDAR) is a major neurotransmitter receptor in the central nervous system (CNS), with functional roles in learning, memory, and sensation. Several mechanisms potentiate NMDARs, and NMDAR hyperexcitability plays pathophysiological roles in many conditions, such as neurodegenerative disease, ischemia, and chronic conditions arising from spinal cord injury. Previous research suggests that the NR2A subunit of the receptor contributes to NMDAR excitotoxicity in heterologous cells and in neurons in vivo. To investigate the role of NR2A in NMDAR excitotoxicity, we have developed a system based on flow cytometry that allows rapid evaluation of the effect of antisense constructs on protein expression and channel function. The enhanced yellow fluorescent protein (EYFP) was fused to obligatory NMDAR subunits, allowing expression to be monitored in living cultured cells. An NR2A antisense construct, asNR2A, specifically and effectively reduced NR2A-EYFP expression. NR1 and NR2A fusion proteins formed functional, excitotoxic channels upon co-expression. The asNR2A RNA significantly reduced NMDAR excitotoxicity when NR2A levels were limiting for channel formation. Using our assay system, further optimization can be achieved rapidly. The asNR2A construct and the assays developed for this study can be used to provide insights into NMDAR biology and disease.

Animals↗

PubMed2005

Tag-array based HPV genotyping by competitive hybridization and extension.

A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (<2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

Alleles↗

PubMed2005

High-throughput screening for kinase inhibitors.

Following G protein-coupled receptors (GPCRs), protein kinases have become the second most important class of targets for drug discovery over the last 20 years. While only four kinase inhibitors have reached the market to date (Fasudil for rho-dependent kinase, Rapamycin for TOR, Gleevec for BCR-Abl, and Iressa for EGFR), many more are already in clinical development. A historical overview of kinase inhibitors was recently published by Cohen. [1] After the previous successes, protein kinases are now regarded as attractive, well-drugable targets, and the analysis of the human genome has yielded 518 protein kinases. [2] We can thus expect screening for protein kinase inhibitors to become even more important in the future. In this review we will focus on the early steps of drug discovery programs producing new lead compounds. We will guide the reader through efficient state-of-the-art assay development and high-throughput screening of large chemical libraries for protein kinase inhibitors.

Drug Evaluation, Preclinical↗

PubMed1995

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia.

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.

Adult↗

PubMed1992

An indirect ELISA for urinary gonadotropins using immobilized human menopausal gonadotropin.

An indirect ELISA for the estimation of urinary gonadotropins is described. Human menopausal gonadotropin is adsorbed on a microtitre plate, where it serves as an immunosorbent. The residual antigonadotropin antibody is captured by the immunosorbent after reaction with the sample or standard and detected with enzyme-labelled antispecies antibody (antirabbit gamma-globulin-horse radish peroxidase). The assay developed here is rapid and satisfies usual validatory criteria expected from an immunoassay. Moreover, it obviates the need for extraction of samples with acetone, as shown by the close agreement between the respective lutropin or follitropin concentrations in extracted and unextracted urine samples.

Animals↗

PubMed2006

A novel method for patch-clamp automation.

An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1-1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated alpha(7) nicotinic receptor ion channels. The system throughput was a single concentration-response curve every 30-40 min or 12-17 6-point concentration-response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.

Animals↗

PubMed1989

The measurement of hyaluronidase activity in human spermatozoa by substrate slide assay and its clinical application.

Acrosomal hyaluronidase activity of individual sperm can be detected by a halo formation around the sperm head on hyaluronic acid substrate slides. The following results were obtained by this method in clinical practice. A significant correlation was found between hyaluronidase activity and sperm concentration in male infertility. Hyaluronidase activity increased as the concentration of sperm increased, and the least hyaluronidase activity was determined at less than 10 X 10(6)/ml. The group with more than 40% motility had a higher hyaluronidase activity than other groups with poor motility. Although there was no significant correlation between hyaluronidase and cumulus dispersion and fertilization rates in immature oocytes, there was an excellent correlation in mature oocytes in human in vitro fertilization. These findings suggest that a newly developed assay will be useful for evaluating sperm fertilizing capacity.

Acrosome↗

PubMed2005

A prospective clinical study of Epstein-Barr virus and host interactions during acute infectious mononucleosis.

BACKGROUND: Characterizing virus-host interactions during self-limited infectious mononucleosis could explain how Epstein-Barr virus (EBV) replication is normally controlled and provide insight into why certain immunocompromised patients fail to contain it. METHODS: University students had an average of 7 clinical and virologic evaluations during acute infectious mononucleosis. EBV was quantified in 697 samples of oral wash fluid, whole blood, peripheral blood mononuclear cells (PBMCs), and plasma by a real-time (TaqMan) polymerase chain reaction (qEBV) assay developed in our laboratory. RESULTS: Twenty of 25 subjects had serologically confirmed primary EBV infection. EBV was cleared from whole blood by a first-order process with a median half-life of 3 days, and its quantity was associated with severity of illness (r2=0.82). Oral shedding persisted at a median of >or=1x104 copies/mL for 32 weeks and was unrelated to severity of illness. Subjects with nonprimary EBV infection shed virus intermittently, and median quantities for all samples became undetectable within 4 weeks. CONCLUSIONS: Using a novel qEBV assay, we demonstrated that young adults with primary EBV infection rapidly cleared virus from blood but not from the oropharynx. High oral concentrations of EBV in asymptomatic persons who have resumed normal activities support the concept that infectious mononucleosis is most likely acquired by kissing.

Adolescent↗