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PubMed1986

Immunoturbidimetric assays of apolipoproteins A, AI, AII, and B in serum.

In these immunoturbidimetric assays developed for apolipoproteins A, AI, AII, and B we used the Cobas-Bio centrifugal analyzer and commercially available antisera and calibrators. Within-day and within-run precision ranged from 1.0 to 5.8% for the four assays. The addition of bilirubin, up to 150 mg/L, or hemoglobin, 50 g/L, to sera did not interfere with the measurement of these apolipoproteins. Comparison of results of the immunoturbidimetric assays with those of radial immunodiffusion assays yielded slopes of 1.002, 0.822, and 0.957 and intercepts of -0.02, 0.47, and 0.03 g/L for apolipoproteins B, A, and AI, respectively. Reference intervals for the four apolipoproteins were determined by using sera from 193 apparently healthy individuals.

Apolipoprotein A-I↗

PubMed1996

Seroepidemiology and control of genital herpes: the value of type specific antibodies to herpes simplex virus.

Herpes simplex virus (HSV) type 2 is the main cause of genital herpes infections. Conventional serological techniques cannot discriminate between antibodies specific to HSV types 1 and 2, so these methods cannot be used in the seroepidemiological investigation of genital herpes infections. The discovery of type specific antigens in HSV-1 and HSV-2, most notably glycoprotein G (gG), has been successfully exploited to develop assays for the detection of type specific HSV antibody. The presence of type specific antibodies provides evidence for infection with HSV-1, HSV-2, or both. Tests which detect humoral responses to HSV-2 have been used during the past decade to investigate populations in various countries. Most surveys of general adult populations in the developed world have found a relatively low seroprevalence of HSV-2, although this does differ between countries. Studies of HSV-2 antibody in developing countries have revealed higher rates of infection in adults. People attending sexually transmitted diseases (STD) clinics have higher rates of HSV-2 infection than general populations sampled in the same country. Assays of HSV type specific antibodies have been used in seroepidemiological surveys that include information about demography and sexual behaviour to identify risk factors for HSV-2 infection. The surveys show that people with a large number of sexual partners are at increased risk of acquiring HSV-2 infection and that a high proportion of HSV-2 infections are asymptomatic. Assays of HSV type specific antibody have been used to show that the presence of pre-existing HSV-1 antibody may reduce the severity of genital HSV-2 infection. Such assays have also been used in studies of the transmission of genital herpes. Proposed interventions to counter the spread of genital herpes include targeted education and vaccination programmes. Assays of HSV type specific antibodies may be used to monitor the effectiveness of such policies.

Adult↗

PubMed2003

A homogeneous, nonradioactive high-throughput fluorogenic protein kinase assay.

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective. Here we present a novel, rapid, robust assay to measure the enzyme activity of low concentrations of several serine/threonine and tyrosine protein kinases. It is based on the use of fluorogenic peptide substrates (Rhodamine 110, bis peptide amide) that are cleaved before phosphorylation to release the free Rhodamine 110; upon phosphorylation, cleavage is hindered, and the compound remains as a nonfluorescent peptide conjugate. The assay can be carried out in single- as well as multiwell plate formats such as 96- and 384-well plates. The signal-to-noise ratio is very high (40), the Z(') is over 0.8, and the signal is stable for at least 4h. Finally, the assay is easily adapted to a robotic system for drug discovery programs targeting protein kinases.

Adenosine Triphosphate↗

PubMed2006

High-throughput screening for modulators of stem cell differentiation.

Realizing the potential of stem cell biology requires the modulation of self-renewal and differentiation, both of which are incompletely understood. This chapter describes methods for the design, development, and implementation of cell-based screens of small molecules, genes and expressed proteins for modulation of stem and progenitor cell fate. These include the engineering of embryonic and other stem cells with gene promoter-reporter protein constructs and their application in automated screening. We discuss considerations of promoter reporter selection, assay development and implementation, and image acquisition, analysis, and data handling. Such black-box screens are useful for the identification of probes of developmental processes and should provide tools that will identify druggable targets for biochemical assays.

Animals↗

PubMed2006

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction.

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.

Aminacrine↗

PubMed2004

SpeedScreen: The "missing link" between genomics and lead discovery.

SpeedScreen is a novel, label-free, in-solution, affinity-based selection methodology for high-throughput screening (HTS) developed at Novartis Pharma. The SpeedScreen protocol comprises in-solution affinity selection, followed by size exclusion chromatography in combination with microbore-liquid-chromatography/electrospray-ionization mass spectrometry (micro-LC/ESI-MS). The authors describe the basic concept behind assay development, HTS, and data analysis with the SpeedScreen technology. Advantages and limitations of SpeedScreen compared to alternative screening technologies are discussed, and an example is given from a SpeedScreen campaign applying this innovative affinity selection concept in HTS.

Chromatography, Gel↗

PubMed2005

Use of a restriction endonuclease cytotoxicity assay to identify inducible GAL1 promoter variants with reduced basal activity.

Inducible promoter fusions are commonly employed to study the biological functions of genes as well as to investigate mechanisms of transcription regulation. A concern for many studies of heterologous gene expression is that steady state transcription may be too high under non-inducing conditions, producing undesired phenotypes prior to induction. Fusions containing the galactose-inducible GAL1 promoter joined to PvuII, a bacterial DNA endonuclease gene, are toxic to yeast cells even under non-inducing conditions, i.e., in glucose media. This toxicity was utilized in conjunction with PCR-based mutagenesis of the GAL1 regulatory region to isolate mutant promoters that retained high inducibility but exhibited reduced basal level expression. The Mig1 repressor binding and putative TATA box regions were unchanged among four mutant promoters examined in detail. However, each promoter contained one or more mutations within previously identified binding sites for the Gal4 activator protein. Genetic assays developed to monitor GAL1p::I-SceI endonuclease-induced recombination demonstrated that basal expression from two of the new promoters (designated GAL1-V4 and GAL1-V10) was strongly reduced. These experiments and additional quantitative luciferase reporter gene assays demonstrate the utility of the approach for identifying promoters that permit more tightly controlled gene expression.

Base Sequence↗

PubMed2006

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Animals↗

PubMed2002

Comparative study of different enzyme immunoassays for measurement of IgM and IgA rheumatoid factors.

OBJECTIVE: To compare the value of various IgM and IgA rheumatoid factor (RF) tests for the diagnosis of rheumatoid arthritis (RA). METHODS: Firstly, the latex test, one global assay (for IgM, IgA, and IgG RF), six IgM, and four IgA RF assays were compared in a particularly challenging situation-that is, with 67 patients with RA, many of whom were latex negative, and 91 non-RA controls, many of whom were latex positive. More detailed evaluation followed with three IgM RF tests (two commercially available kits and one assay developed in our laboratory) and two IgA RF tests (one commercially available and one from our laboratory) in two more representative samples of rheumatological patients (146 RA and 75 non-RA controls). RESULTS: Diagnostic performance differed considerably between the assays. For IgM RF detection the highest sensitivity (88%) was obtained with the Diamedix kit (specificity 67%) and for IgA RF with the Inova kit (sensitivity 65%, specificity 88%). Combining one IgM and one IgA RF test improved diagnostic performance when both tests were in agreement, but at the cost of yielding 15-27% of discrepant results which did not help in ruling RA in or out. Mean concentration values differed significantly among IgM RF tests, and in most cases concentrations were not correlated. CONCLUSIONS: Available tests for IgM RF isotype vary in accuracy, and none is uniformly better than all the others. For IgA RF isotype, the Inova kit appears to be the best. Quantitative results cannot be compared across tests. Combination of one IgM and one IgA RF test may improve diagnostic accuracy.

Adult↗

PubMed2004

FIZICS: fluorescent imaging zone identification system, a novel macro imaging system.

Constantly improving biological assay development continues to drive technological requirements. Recently, a specification was defined for capturing white light and fluorescent images of agar plates ranging in size from the NUNC Omni tray (96-well footprint, 128 x 85 mm) to the NUNC Bio Assay Dish (245 x 245 mm). An evaluation of commercially available products failed to identify any system capable of fluorescent macroimaging with discrete wavelength selection. To address the lack of a commercially available system, a custom imaging system was designed and constructed. This system provides the same capabilities of many commercially available systems with the added ability to fluorescently image up to a 245 x 245 mm area using wavelengths in the visible light spectrum.

Biological Assay↗

PubMed2002

The bacterial cell wall as a source of antibacterial targets.

This review focuses on target-based approaches for developing new chemical classes of antibacterial agents aimed at the bacterial cell wall. The clinical success of antibiotics such as beta-lactams and glycopeptides validates this chemotherapeutic strategy and emerging resistance to these agents warrants the development of new antibacterial drugs. Understanding the mechanism of action and resistance to beta-lactams and glycopeptides at a molecular level has supported the development of new agents that prevent transpeptidation and transglycosylation reactions of peptidoglycan polymerisation. The enzymes involved in the synthesis of the peptidoglycan structural unit have also been targeted for antibacterial discovery. The influence of bacterial genetics and genomics, structural biology, assay development and the properties of known inhibitors on these approaches will be discussed in the context of drug discovery.

Anti-Bacterial Agents↗

PubMed2003

Proof of principle: detection of genotoxicity by a fluorescence-based recombination test in mammalian cells.

Genotoxicity tests available today have several shortcomings. The widely applied Ames assay measures mutations in bacteria, thereby disregarding the physiological particularities of the human cell and organism.We provide first evidence for a new concept of genotoxicity detection in living human cell cultures. The data were obtained by use of a newly developed assay, which is based on the quantification of fluorescent signals, i.e. counting of the relative number of fluorescent cells in the sample. It is characterised by a short reaction time and fulfills the requirements for automated performance. The new system monitors chromosomal rearrangements and, therefore, is predicted to detect a broad spectrum of genotoxic substances. Indeed, we demonstrate the genotoxic effect of ionising radiation, of an Ames assay positive compound, and of two compounds which are poorly mutagenic in the Ames assay. The new assay will be optimised further and adapted to the requirements for routine analysis in order to help to further reduce animal experimentation in genotoxicity testing.

Animal Testing Alternatives↗

PubMed2006

Training technologists for the genomic age.

Molecular techniques are playing an ever-increasing role in all areas of anatomic and clinical pathology. The field is currently in need of well-trained technologists in this area of the clinical laboratory who are situated to bridge the current state of practice and the continuing developments in high complexity testing. For the close term, use of "home-brew" and analyte specific reagents (ASR)-based tests will require well-trained personnel with strong biomedical science backgrounds and a thorough understanding of technologies used in assay development. Here, we discuss the selection and evaluation of molecular diagnostic training preceptor sites and tasks indicated for trainees that most meet the needs of the newest facet of the laboratory. We present evaluation tools developed over the course of four years of clinical education used to assess practical performance of trainees in a molecular diagnostic pathology laboratory and conclude with considerations for future training of laboratory technologists.

Clinical Laboratory Techniques↗

PubMed1991

Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens.

cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.

Cloning, Molecular↗

PubMed1989

The initiator tRNA acceptance assay as a short-term test for carcinogens. 5. Results with 42 cytostatic drugs.

The activity of 42 cytostatic drugs used for the treatment of human cancer was tested by the initiator tRNA acceptance assay for carcinogens. Of 17 drugs carcinogenic for rodents, 16 (94.1%) gave a positive response in the assay and six (85.7%) out of seven non-carcinogens showed no activity. The predictive value of the test for cytostatics was 91.7%. Treatment of tRNA with several cytostatics resulted in an inhibition of its acceptance for L-methionine. Cyclophosphamide, dibromdulcitol, 5-deoxy-5-fluorouridine and vincristine also inhibited, in addition to this, the charging of unfractionated tRNA from rat liver with L-alanine, L-lysine, L-phenylalanine and L-valine. Some drugs apparently react with the same target nucleoside which is common for all species of tRNA (probably the terminal adenosine residue that is esterified with amino acids). Such compounds do not yield reliable results in the initiator tRNA acceptance assay since this inhibitory effect interferes with the stimulating effect characteristic for carcinogens. However, results of the present study agree well with those obtained earlier with different classes of compounds (N-nitroso compounds, mycotoxins, etc.) and indicate that this newly developed assay may be a useful alternative also for the testing of carcinogenicity of cytostatic drugs.

Amino Acids↗

PubMed2004

Hormonal therapies and osteoporosis.

Osteoporosis, now defined as a disease characterized by low bone mass and a microarchitectural deterioration of bone tissue leading to enhanced bone fragility and fracture risk, is a major public health problem. Classic hormonal therapies to prevent and treat osteoporosis associated with menopause have recently been questioned due to the risk/benefit ratio of prolonged treatment. There is a critical need for safe and effective alternative therapeutics for this disease. Nonhuman primates have been used as models to assess bone changes associated with estrogen deficiency because their trabecular and cortical bone remodeling processes, monthly menstrual cycles, and reproductive-hormone patterns are similar to those of humans. The ovariectomized nonhuman primate has become the preferred model in which to study effects on bone remodeling, particularly with regard to bone mass, architecture, and strength, in fulfillment of studies required by international guidelines for the development of antiosteoporotic drugs. The nonhuman primate is amenable to several methodologies that assess bone quantity and quality, including dual energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT), histology, static and dynamic histomorphometry, and biomechanical testing, as well as assays developed for clinical use, which serve as biomarkers of bone metabolic processes. The use of the nonhuman primate model in the assessment of osteoporosis therapeutics, both hormonal (sex steroids and their analogues, parathyroid hormone) and nonhormonal (bisphosphonates), has provided valuable information on the safety and efficacy as well as the mechanisms of bone loss associated with estrogen deficiency that is directly applicable to the human situation.

Animals↗

PubMed2006

High-throughput screening: update on practices and success.

High-throughput screening (HTS) has become an important part of drug discovery at most pharmaceutical and many biotechnology companies worldwide, and use of HTS technologies is expanding into new areas. Target validation, assay development, secondary screening, ADME/Tox, and lead optimization are among the areas in which there is an increasing use of HTS technologies. It is becoming fully integrated within drug discovery, both upstream and downstream, which includes increasing use of cell-based assays and high-content screening (HCS) technologies to achieve more physiologically relevant results and to find higher quality leads. In addition, HTS laboratories are continually evaluating new technologies as they struggle to increase their success rate for finding drug candidates. The material in this article is based on a 900-page HTS industry report involving 54 HTS directors representing 58 HTS laboratories and 34 suppliers.

Cells↗

PubMed1994

GDP-ribosyl cyclase activity as a measure of CD38 induction by retinoic acid in HL-60 cells.

Retinoic acid (RA) treatment of HL-60 cells induces surface expression of CD38. This lymphocytic antigen is also a novel bifunctional enzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca2+ mobilizing metabolite of NAD+. The synthetic activity of CD38 is very difficult to detect because of the concurrent hydrolytic activity. In this study, a Ca2+ release assay capable of detecting submicromolar concentrations of cADPR was used to demonstrate the induction of ADP-ribosyl cyclase activity in HL-60 cells by RA. Concomitantly, cADPR hydrolase activity was also increased. The results were further substantiated by using a newly developed assay for GDP-ribosyl cyclase activity. This assay uses NGD+ as substrate instead of NAD+. The resulting fluorescent product, cyclic GDP-ribose, is resistant to hydrolysis and accumulates, making it a highly sensitive and convenient assay for CD38-like enzymes.

ADP-ribosyl Cyclase↗