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At least 811 records · Page 45Linked to original sources

PubMed2005

Remnant lipoproteins and atherosclerosis.

A recently developed assay for quantification of remnant-like particle cholesterol has provided considerable evidence that reinforces the concept that elevated levels of plasma remnants are associated with increased cardiovascular disease in different populations and distinct patient groups. In this review, we provide a brief summary of the most recently published studies, emphasizing the clinical relevance of remnant analysis. We discuss recent evidence that sheds light on the mechanisms that may underlie the atherogenicity of remnant lipoproteins. Taken together, these data provide new insight into the significance of remnant lipoproteins in the onset and development of premature atherosclerosis.

Biomarkers↗

PubMed1999

Biochemical assay for amyloid beta deposits to distinguish Alzheimer's disease from other dementias.

Biochemical markers for Alzheimer's disease (AD) are of great value for precise diagnosis and in studies of the pathogenetic processes of this disease. A new biochemical assay allowing to differentiate AD from other forms of dementia is described. The assay is based on the extraction of amyloid beta (A beta) from milligram amounts of brain tissue by using 20% acetonitrile in 0.1% trifluoroacetic acid and its detection by Western blotting. The presence of the 4 kDa A beta was demonstrated in all cases of AD (n = 8) that were diagnosed by the independent histopathological examination of the postmortem tissues. No A beta was found in tissue extracts from seven out of eight cases of other forms of dementia. In contrast to other biochemical techniques of A beta detection in brain, the developed assay is simple; it does not require any special equipment and allows detection of A beta using milligram amounts of brain tissue.

Alzheimer Disease↗

PubMed2000

Analytical and prospective evaluation of urinary cytokeratin 19 fragment in bladder cancer.

PURPOSE: We evaluated CYFRA 21-1, an immunoradiometric assay, developed to detect soluble cytokaratin 19 fragment, for its diagnostic performance in bladder transitional cell carcinoma as well as its analytical performance. MATERIALS AND METHODS: We assessed CYFRA 21-1 in the serum and urine of 182 patients, including 66 with bladder transitional cell carcinoma (group 1), 66 with another urological pathology (group 2) and 50 free of urothelial disease (group 3). The power of urinary CYFRA as a diagnostic test for bladder transitional cell carcinoma was evaluated by receiver operating characteristics curve analysis. Analytical performance was assessed by determining intra-assay and interassay precision, and accuracy by dilution testing and recovery of supplemented analyte. RESULTS: Mean urinary CYFRA plus or minus standard deviation was 154.39+/-49.00, 22.6+/-8.9 and 2.40+/-0.14 ng./ml. in groups 1 to 3, respectively (significantly different). Receiver operating characteristics curve analysis revealed optimal 96.9% sensitivity and 67.2% specificity for a threshold value of 4 ng./ml. Analytical determination showed that intra-assay and interassay precision provides a satisfactory coefficient of variation. The assay for accuracy had acceptable recovery in diluted samples as well as in those with supplemented analyte. CONCLUSIONS: The urinary immunoradiometric CYFRA 21-1 assay performs well analytically. Urinary CYFRA 21-1 is a useful marker for diagnosing transitional cell carcinoma and provides sensitivity in low grade disease.

Aged↗

PubMed2000

Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR.

A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa. Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring. This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp. expressing the NDO inducible enzyme. The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml. Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments.

Amino Acid Sequence↗

PubMed1985

[Analysis of chemosensitivity against a human breast cancer xenograft (MX-1) with the subrenal capsule assay in BDF1 mice].

The antitumor activity of 27 anticancer drugs against a human breast cancer tumor (MX-1) has been studied using the subrenal capsule assay developed by A.E. Bogden et al. in 1978. Without immunosuppression with cyclophosphamide, BDF1 mice transplanted with MX-1 were treated with various drugs. The antitumor activity was evaluated by the tumor growth inhibition rate on day 6 after treatment. Among the 27 anticancer drugs tested, 10 compounds (37%) which showed more than 75% tumor growth inhibition rate were considered to be active. On the other hand, 8 compounds out of 27 drugs (30%) which showed less than 50% tumor growth inhibition rate were considered to be inactive. When the antitumor activity between the subrenal capsule assay in BDF1 mice and the subcutaneous transplantation assay in nude mice were compared, both assays were well correlated (r = 0.787, p less than 0.001). These results suggest that the antitumor activity of drugs can be evaluated faster, cheaper and easier by the subrenal capsule assay compared with the subcutaneous transplantation assay in nude mice.

Animals↗

PubMed1985

Cooperative evaluation of human tumor chemosensitivity in the soft-agar assay and its clinical correlations.

In supporting the human-tumor cloning effort of the Southwest Oncology Group, we conducted an independent retrospective study to evaluate the clinical correlations of the soft-agar colony-forming assay developed by Hamburger and Salmon (1977). This study was made with the cooperation of 76 clinicians and 11 hospitals in Greater New Orleans. In a 10-month trial (July 1982 to May 1983), we received 134 human tumors of 26 classifications and achieved 76% success in colony growth from 122 plated samples. Retrospective correlations between the in vitro chemosensitivity of tumor colonies and clinical drug responses were made possible in 31% of the patients. Evaluation of 45 in vitro and in vivo associations indicated a combined sensitivity of 0.65 and a specificity of 0.68 for the assay. Technical refinements and the selectivity of the assay are discussed.

Antineoplastic Agents↗

PubMed1992

Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay.

The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.

Guanidines↗

PubMed2003

Molecular epidemiological survey on the vectors of Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini (Spirurida: Thelaziidae).

A Polymerase Chain Reaction (PCR)- based assay developed for the specific identification of Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini (Nematoda, Spirurida), which cause bovine ocular thelaziosis, was evaluated for its usefulness in detecting the intermediate hosts and in estimating the infection prevalence of vectors in field conditions throughout 5 years (from 1997 to 2001). A total of 5190 flies were captured and identified as Musca larvipara, Musca osiris, Musca autumnalis, Musca tempestiva or Musca domestica. Genomic DNA was extracted from pools constituted by heads, thoraces, abdomens and wings of 10 flies of each species, and 2076 samples were subjected to a PCR assay to specifically detect the ribosomal ITS-1 sequence of bovine Thelazia. Amplicons were sequenced and subjected to digestion with CpoI restriction enzyme. M. autumnalis, M. larvipara, M. osiris and M. domestica species were shown to be PCR positive. T. gulosa was specifically detected by PCR in M. autumnalis, M. larvipara, M. osiris and M. domestica, whereas T. rhodesi is in M. autumnalis and M. larvipara. Of 27 positive samples, 23 were positive for T. gulosa and 4 for T. rhodesi, with a mean prevalence of 2.86% in the whole fly population collected. The highest mean prevalence values of infection were detected in M. autumnalis (4.46%) and M. larvipara (3.21%), and the former species was confirmed to be the vector of T. gulosa and T. rhodesi. This study is the first report of M. osiris as a vector of T. gulosa and M. larvipara as a vector of T. gulosa and T. rhodesi under natural conditions. The occurrence of Thelazia in fly populations in the Apulia region of Italy (in the 5 grazing seasons considered) indicates that cattle thelaziosis is enzootic in southern Italy. This molecular assay should be a useful epidemiological tool for assessing the role of different species of flies as intermediate hosts of thelaziae.

Animals↗

PubMed2002

The National Toxicology Program rodent bioassay: designs, interpretations, and scientific contributions.

The National Toxicology Program rodent cancer bioassay program design evolved from that of the National Cancer Institute in the 1970s. Groups of 50 or more mice are assigned to control or treatment groups. Test substances are given at three dose levels by intubation, dietary or drinking water consumption, or dermal or inhalation exposure. Dosing starts at age 5-6 weeks and lasts for 2 years, when surviving animals receive a complete histopathologic examination. Statistical approaches accommodate survival differences and no longer require differentiation between fatal and incidental tumors. Photocarcinogenicity studies, employing SKH-1 hairless mice, evaluate onset of skin papillomas and incidences at 1 year. Top doses are chosen to expose animals to a minimally toxic challenge and lower doses to operate within the linear range of kinetics. This dosing allows comparison of results across studies. Bioassay and ancillary studies successfully identify tumor-causing agents in rodents, provide information on dose-response, and characterize other chemical-related toxicities. NTP and Ramazzini Foundation bioassay designs differ in several aspects, but bioassays at both institutions provide chemical-specific information for predicting human carcinogens, thus providing for protection of public health. Bioassays constitute an essential information reference set for new assay development and further investigations into mechanisms of action. The scientific community and the public owe a huge debt of gratitude to Dr. Cesare Maltoni of the European Foundation of Oncology and Environmental Sciences and to Dr. David P. Rall of the National Institute of Environmental Health Sciences for their foresight and wisdom in creating and nurturing these bioassay programs.

Animals↗

PubMed1995

Triple-label hybridization assay for type-1 diabetes-related HLA alleles.

We describe a method for the detection of two type 1 (insulin-dependent) diabetes susceptibility (*0201, *0302) alleles and two protective (*0301, *0602/0603) alleles of the HLA-DQB1 gene on the human major histocompatibility complex (MHC). The test is based on DNA amplification with PCR followed by simultaneous, allele-specific triple-label hybridization performed in microtitration wells. In the hybridization, very short allele-specific oligonucleotides labeled with europium (Eu), terbium (Tb) or samarium (Sm) are used. The labeled probes could be detected using time-resolved fluorometry with sensitivities of 1 x 10(7), 3 x 10(8) and 3 x 10(8) molecules, respectively. Cross-reactions were not found among samples containing 14 common DQB1 alleles. To test the utility of the developed assay, 100 DNA and 14 dried blood spot samples with known DQB1 alleles were analyzed. A 100% agreement with the reference method was reached. Thus, this triple-label hybridization assay proved to be suitable even for detection of a large number of samples.

Alleles↗

PubMed1989

Cell receptor assays.

There are four general assay methods used to quantify a drug/biologic in a preparation, including: (1) in vivo bioassays; (2) in vitro bioassays; (3) immunoassays; and (4) receptor assays. The cell receptor assay is used to evaluate the first step in the molecular action of the drug biologic, its interaction with a specific cellular receptor. Subsequently, the drug biologic must initiate other events, such as internalisation, signal transduction, and/or alterations of one or more cellular constituents in order to elicit its biological effect. Major factors to consider in cell receptor assay development include: (1) establishment of a reference standard preparation; (2) labelling; purifying and characterisation of the biologic drug; (3) cell receptor source; (4) methodology, e.g. separation of bound and free, and other factors affecting accuracy and reproducibility; (5) ligand specificity; and (6) correlation with bioactivity. It should be emphasised that cell receptor binding cannot be assumed to correlate with biological activity because of the requirement that subsequent steps must take place prior to achieving the final response. Chemically altered drugs biologics may bind to a specific cell receptor without eliciting a biological activity. Thus, utilisation of a cell receptor assay requires careful evaluation at both the chemical and biological levels prior to its acceptance as a measure of potency.

Animals↗

PubMed2004

PGD for autosomal dominant polycystic kidney disease type 1.

Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children.

Adult↗

PubMed2004

Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings.

There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings. In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Perú which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth. MODS detected 94.0% of 1,908 positive sputum cultures, whereas Lowenstein-Jensen (LJ) culture detected only 86.9% (P < 0.001). The median time to culture positivity was 8 days (compared to 16 days for the same 208 samples by LJ culture; P < 0.001, Wilcoxon signed rank test). The results obtained by direct susceptibility testing using MODS demonstrated excellent concordance for isoniazid and rifampin and the detection of multidrug resistance with those obtained by indirect colorimetric methods: the microplate Alamar Blue assay (MABA) and the tetrazolium microplate assay (TEMA) (agreement, 95, 98, and 94%; kappa values, 0.8, 0.7, and 0.7, respectively). The concordance of the susceptibility testing results for ethambutol and streptomycin was poor. MODS is a novel assay which can detect the organisms responsible for TB and MDR-TB directly from sputum inexpensively, rapidly, and effectively. A comprehensive prospective evaluation of MODS is under way in Perú, and independent validation in nonresearch laboratories should be undertaken at the earliest opportunity.

Antitubercular Agents↗

PubMed1988

Development of a lymphocyte transformation assay for rotavirus in whole blood and breast milk.

A multivariate assay was developed for detecting lymphoproliferative responses to rotavirus in whole blood and breast milk. Detection of lymphocyte transformation in samples from healthy uninfected adults required an assay incorporating a large number of antigen and cell concentrations and several incubation periods before pulsing. In contrast, lymphoproliferation to rotavirus was observed over a wider range of these variables in blood collected from a kidney recipient nine days post-rotavirus infection. Lymphoproliferation to rotavirus was detected in 32.5% of breast milk samples tested. The magnitude of lymphoproliferation to rotavirus did not correlate with rotavirus-specific IgG ELISA antibody titres in sera or with rotavirus-specific IgA ELISA antibody titres in breast milk.

Adult↗

PubMed1991

Physiological and pathological influences on sheep blood plasma amine oxidase: effect of pregnancy and experimental alloxan-induced diabetes mellitus.

A copper-containing amine oxidase is present in sheep blood plasma and has a high capacity to deaminate spermine and spermidine. The physiological function of this enzyme remains to be determined. Sheep blood plasma amine oxidase (SPAO) was measured by its ability to deaminate spermidine (700 microM) using a peroxidase-linked colorimetric assay developed for microtitre plates. SPAO activity has been studied in a group of Welsh Mountain sheep with experimental alloxan-induced diabetes. This resulted in an increase in SPAO activity which reached a peak of 70 days after alloxan treatment (60 per cent increase). This change could be seen in both pregnant and non-pregnant diabetic sheep. In normal pregnant ewes, SPAO activity remained stable for the first 100 days of pregnancy but declined by 50 per cent in the last month of pregnancy. Together, these findings suggest that SPAO activity is controlled by hormonal influences. This sensitive and convenient assay method could provide clues as to the physiological significance of SPAO and may be a useful clinical chemical indicator in the sheep.

Amine Oxidase (Copper-Containing)↗

PubMed1999

Investigation of seroprevalence of respiratory virus infections in an infant population with a multiantigen fluorescence immunoassay using heel-prick blood samples collected on filter paper.

Respiratory viruses are an extremely common cause of childhood morbidity. However, the current seroprevalence of viruses in infant populations is difficult to establish because invasive venipuncture may be technically and ethically unacceptable. This prospective study aimed to establish the seroprevalence of respiratory viruses in an infant population by use of a novel multiantigen fluorescence immunoassay against common respiratory viruses, using heel-prick blood samples collected on filter paper. Mothers and babies were recruited in the immediate peripartum period in the Royal Maternity Hospital, Belfast. Cord blood samples at birth and heel-prick filter paper blood samples at 7 mo were collected for measurement of virus-specific IgG to respiratory syncytial virus, influenza A virus, adenovirus, and parainfluenza virus type 1, type 2, and type 3 by indirect immunofluorescence using a multiviral assay developed for this purpose. Of 386 mothers approached, 325 (84%) permitted follow-up at 7 mo, and of these, 256 (79%) agreed to the heel prick. From 234 paired samples, 125 infections were documented. Adenovirus infections were commonest, 53 (22.6%), followed by respiratory syncytial virus, 32 (13.7%); influenza A virus, 22 (9.4%); parainfluenza virus type 3, 14 (6%); parainfluenza virus type 1, 2 (0.85%); and parainfluenza virus type 2, 2 (0.85%). These results demonstrate the seroprevalence of a range of respiratory viruses in an infant population, using a novel multiviral immunoassay. The filter paper collection of blood samples and multiantigen assay format has implications for easy, widespread viral serodiagnosis in both seroepidemiology studies and in the diagnosis of pediatric viral illnesses. Filter paper permits recovery of respiratory virus-specific IgG and can be used as a simple and acceptable epidemiologic and diagnostic tool.

Antigens, Viral↗

PubMed2000

Evaluation of a bead-based enzyme immunoassay for the rapid detection of osteocalcin in human serum.

BACKGROUND: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin. METHODS: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS((R)) EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH(2) terminus. RESULTS: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 microg/L and a linear dose-response curve between 4.5 and 100 microg/L. The intraassay imprecision (CV) was 5-8% (n = 21); the interassay variation was 6-9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays. CONCLUSIONS: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.

Adult↗

PubMed1999

Development and validation of species-specific nested PCRs for diagnosis of acute sarcocystiosis in sheep.

Sheep may be infected by four species of Sarcocystis. Two of these species, Sarcocystis tenella and Sarcocystis arieticanis, are pathogenic. They may cause abortion or acute disease during the early phase of infection, and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis in sheep has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic species. In addition, most of these tests show a reasonable sensitivity only for the late phase of infection. Therefore, diagnosis of acute sarcocystiosis has been based mainly on post-mortem examination, i.e. after the animal had succumbed to the disease. Here we established species-specific nested PCR assays based on unique small subunit ribosomal RNA gene sequences of S. tenella and S. arieticanis. These PCR assays specifically detect DNA of the homologous species in blood samples of sheep. No cross-reactions were observed with the heterologous pathogenic species, the non-pathogenic species Sarcocystis gigantea, or the closely related coccidia Toxoplasma gondii and Neospora caninum. In sheep experimentally infected with S. tenella or S. arieticanis, positive PCR results were correlated with the early phases of multiplication (endopolygeny) of the parasites. By contrast, Sarcocystis-specific antibodies were detected by an enzyme-linked immunosorbent assay only during the terminal phase of endopolygeny or thereafter. Thus, the nested PCR assays developed here enable, for the first time, the diagnosis and differentiation of infections with S. tenella and S. arieticanis in living sheep during the acute phase of the disease and facilitate comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.

Acute Disease↗