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PubMed2004

NMP-22 for bladder cancer screening and surveillance.

In 2004, more than 56,000 Americans will be diagnosed with bladder cancer (American Cancer Society, 2004). Urethrocystoscopy is considered the gold standard for bladder cancer diagnosis, but it is not a screening test. Researchers have been challenged to develop a noninvasive urine test that reliably differentiates hematuria associated with bladder cancer from that associated with nonmalignant etiologies. Several well-established and some novel tests for bladder cancer screening diagnosis and surveillance, including urinalysis, voided cytology, and the Nuclear Matrix Protein 22 (NMP-22) assay, one of a growing number of assays developed as a specific, sensitive noninvasive test for bladder cancer, are reviewed. Until the sensitivity of the NMP-22 is improved, or another urine assay emerges that provides significantly greater sensitivity while maintaining adequate specificity, urethrocystoscopy will remain the reference test for detecting and diagnosing bladder cancer and its recurrence.

Biomarkers, Tumor↗

PubMed1987

Induction of ethylenediamine hypersensitivity in the guinea pig and the development of ELISA and lymphocyte blastogenesis techniques for its characterization.

Ethylenediamine (EDA) is reported to be a poorly characterized iatrogenic and occupational contact sensitizer. To better characterize EDA hypersensitivity, a guinea pig model was employed in which the animals were exposed epicutaneously to simulate conditions of human exposure, and selected immune parameters were measured. Induction of hypersensitivity was by the Buehler occluded patch method (6 hr application/day, once a week for 3 consecutive weeks) to 10, 20, 30, or 40% EDA, using either an ethanol or acetone/corn oil vehicle. Fourteen days after the last induction, guinea pigs were challenged by patch application of 2% EDA (nonirritating). The incidence of responders for erythema in the 10% EDA (ethanol) treatment group was 83 and 50% at 24 and 48 hr, respectively. In the 10% EDA (acetone/corn oil) group the corresponding values were 50 and 17%. For 20, 30, and 40% EDA, in either vehicle, the incidence of erythema was 83 to 100%. Severity grades (scale = 0-3) for cutaneous reactions to increasing concentrations of EDA in ethanol ranged from 0.8 to 2.5; those for EDA in acetone/corn oil ranged from 0.6 to 2.8. Using an enzyme-linked immunosorbent assay developed to detect the predominant serum antibodies to EDA, it was shown that guinea pigs treated by patch application did not produce the main allergic antibody IgG specific for EDA. However, intradermal administration of an EDA-guinea pig serum albumin conjugate (EDA-GSA) to guinea pigs presensitized by patch application resulted in antibody production by 39 and 86% of the animals, at the initial and second dosing, respectively. An in vitro blastogenesis assay, using peripheral blood lymphocytes from EDA-sensitized guinea pigs, was developed to identify specific chemical allergens implicated in vivo sensitization. Maximum tritiated thymidine ([3H]TdR) incorporation by lymphocytes stimulated in vitro with EDA-GSA was observed on Day 7. Optimal antigen concentration for maximum lymphocyte proliferation ranged from 5 to 50 micrograms/ml, the major variation being attributable to interanimal differences. These results indicate that epicutaneous application of EDA in the guinea pig induces a Type IV delayed hypersensitivity; immunological memory to the hapten is maintained in cultured lymphocytes, suggesting the potential usefulness of the lymphocyte transformation test for in vitro diagnosis of chemically induced hypersensitivity in humans.

Animals↗

PubMed1977

Some immunological effects of penicillamine.

Immunological effects of D- and D,L-penicillamine (PA) were studied in efforts to develop assays for synthetic D or D,L analogs and to contribute to the understanding of the mechanism(s) of action of D-PA in rheumatoid arthritis. At the highest doses tolerated by mice, D,L-PA did not significantly inhibit the development of haemagglutinating antibodies in vivo. In studies in vitro with T lymphocytes, D-PA at 1 mM concentration inhibited both concanavalin A- and phytohaemagglutinin-induced transformation as assayed by [3H]thymidine incorporation, but D-PA concentrations of 5 mM were required to inhibit concanavalin A-induced amino acid uptake. No effect of D-PA was observed either on the induction of cytotoxic T cells or on the attack of specifically sensitized T cells on target cells. It is of interest that D-PA at 1 mM concentration did inhibit lipopolysaccharide-induced transformation, which predominately stimulates B lymphocytes. The effects of PA on the induced transformation of T and B cells deserve further attention for studies with analogs of PA.

Animals↗

PubMed2007

Target validation in drug discovery.

The process of target validation identifies and assesses whether a molecular target merits the development of pharmaceuticals for therapeutic application. The most valuable application of high content screening to target validation is at the early stages of the process when genetic methods (including RNA interference--RNAi) are being applied to many potential targets. At this stage both throughput and indepth analysis are required. This process is illustrated using various examples from the area of oncology target validation. The Akt signal transduction pathway is used to illustrate an efficient way of identifying HCS compatible reagents for use in assay development. RNAi transfection methods are discussed. A description is given of an HCS assay that simultaneously measures two nodes of the Akt pathway: Akt substrate phosphorylation and RPS6 phosphorylation. Another example of an assay measuring proliferation (DNA synthesis) and apoptosis (Histone H2B phosphorylation) within the same cell population is used to illustrate the combination of typical phenotypic assays.

Adaptor Proteins, Signal Transducing↗

PubMed1984

Steroid hormone binding macromolecules in meningiomas. Failure to meet criteria of specific receptors.

Cytoplasmic estrogen and progesterone binding proteins were assayed in 26 meningiomas using an assay developed for the measurement of estrogen and progesterone receptors in human target tissue. The steroid specificity of the binding proteins was studied in both individual and pooled meningioma tissue by competitive binding assays. Eight meningiomas contained significant amounts of estrogen-binding protein and 18 contained significant amounts of progesterone-binding protein. There was no correlation between the amount of hormone-binding protein and patient age, sex, menstrual status or presentation, or tumor occurrence, location, or pathologic features. The competition studies demonstrated a lack of steroid specificity for these hormone-binding proteins. Therefore, the authors conclude that, contrary to recent reports, the hormone-binding proteins found in meningiomas are unlikely to be specific steroid receptors.

Adult↗

PubMed2003

Activity-based proteomics: enzyme chemistry redux.

The principles of enzyme chemistry, mechanism of action and inhibitor design are being applied to proteomics by the development of activity-based probes. This approach suggests a potentially broad method for interrogating enzyme family members, both known and unknown, in cells and proteomic fractions without the need for individual assay development and isolation. The serine hydrolases and cysteine proteases have provided the proofs of concept for activity-based proteomics, and other studies are rapidly following. The result will be a proteomics technology of great value to drug discovery and development.

Enzyme Inhibitors↗

PubMed1999

Spontaneous and photosensitiser-induced DNA single-strand breaks and formamidopyrimidine-DNA glycosylase sensitive sites at nucleotide resolutionin the nuclear and mitochondrial DNA of Saccharomyces cerevisiae.

A system is described for mapping oxidative DNA damage (sites sensitive to formamidopyrimidine-DNA glycosylase and single-strand breaks) at nucleotide resolution in the nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Our 3' end labelling method is sensitive and was first developed using the well-studied inducer of oxidative DNA damage, methylene blue (MB) plus light. We treated yeast DNA in vitro with this so as to maximise levels of damage for assay development. Unfortunately, MB does not remain in yeast cells and yeast DNA repair mutants sensitive to active oxygen species are not sensitive to this agent, thus for in vivo experiments we turned to a polycyclic aromatic, RO 19-8022 (RO). This resulted in oxidative DNA damage when light was applied to yeast cells in its presence. The spectra of enzyme-sensitive sites and single-strand breaks induced by MB in vitro or by RO plus light in vivo or in vitro were examined in two yeast reporter genes: the nuclear MFA2 and the mitochondrial OLI1. The experiments revealed that most of the enzyme-sensitive sites and single-strand breaks induced by MB or RO plus light are at the same positions in these sequences, and that these are guanines.

Binding Sites↗

PubMed2002

Use of epitope mapping to identify a PCR template for protein amplification and detection by enzyme-linked immunosorbent assay of bovine herpesvirus type 1 glycoprotein D.

Infection with bovine herpesvirus type 1 (BHV-1) occurs worldwide and causes serious economic losses due to the deaths of animals, abortions, decreased milk production, and loss of body weight. BHV-1 is frequently found in bovine semen and is transmitted through natural service and artificial insemination. The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. In the present study, ordered deletions of the full-length BHV-1 glycoprotein open reading frame were used to identify an epitope recognized by a specific monoclonal antibody (MAb). A glycoprotein D fragment containing this epitope was then amplified using an in vitro protein amplification assay developed previously (J. Zhou, J. Lyaku, R. A. Fredrickson, and F. S. Kibenge, J. Virol. Methods 79:181-189, 1999), and the resulting peptide was detected by indirect enzyme-linked immunosorbent assay (ELISA) with the specific MAb. This method detected 0.0395 50% tissue culture infective dose of BHV-1 in raw bovine semen, which was 1,000-fold more sensitive than traditional PCR. We therefore conclude that this in vitro protein amplification assay combined with ELISA has superior sensitivity for direct virus detection in clinical samples.

Animals↗

PubMed1994

Prevention of side effects by hemoglobin solutions; the selection of optimal test models, especially concerning thrombogenicity.

Modification of hemoglobin (Hb) by crosslinking and polymerization results in an improved oxygen release capacity and a prolonged vascular retention time. Modification improves the efficacy and prevents certain side effects. It eliminates leakage of Hb through the kidneys and accumulation in the tubuli. Another important issue is the degree of purification of Hb solutions. Traces of membrane fragments may cause immunogenic and thrombogenic side effects. To determine the contamination with erythrocyte membrane fragments, we developed assays for glycophorin-alpha and phospholipids. Special models were evaluated for testing the maximum allowable level of membrane contamination. As an in vitro model for thrombogenicity we used confluent monolayers of human umbilical vein endothelial cells. These cells were incubated with Hb solutions and subsequently tested on tissue factor (TF) procoagulant activity. TF was tested by the factor VII-catalyzed activation of factor X. The lower detection limit of this assay for endotoxin was 0.5 ng/ml. Hb did not cause any tissue factor expression even after prolonged incubation. No cooperation was found within endotoxin. As an in vivo test on thrombogenicity we developed a guinea pig model in which we can follow the generation of fibrinopeptide A (FPA). This is one of the most sensitive markers for thrombin activation in vivo. When slightly contaminated Hb solutions (phospholipid content 2 nmol/ml) were infused in the presence of factor Xa at a dose (9 micrograms/kg) which in itself did not induce FPA generation, we observed an increase in FPA levels in the plasma from 1.2 +/- 0.4 ng/ml to 5.2 +/- 0.7 ng/ml. Factor Xa is used to mimic a stressed clinical condition with activated coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Kinetic studies of small molecule interactions with protein kinases using biosensor technology.

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.

Binding Sites↗

PubMed1997

Recent advances in diagnosis of paragonimiasis.

Paragonimiasis in endemic areas can be diagnosed by clinical symptoms. However, the diagnosis should always be confirmed by microscopic examination of the sputum or stool in order to find Paragonimus eggs. Within recent years marked advances in diagnosis of paragonimiasis have been made. Two new approaches comprising a genetic probe and immunological tests have been developed with claims to be as good or better than microscopic examinations. This report reviews these two areas, especially in paragonimiasis caused by Paragonimus heterotremus and P. westermani. In addition, problem areas in assay development are discussed.

Animals↗

PubMed2004

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction.

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.

Biological Assay↗

PubMed1999

A comparison of performances of four enzymes used in ELISA with special reference to beta-lactamase.

Horse radish peroxidase, alkaline phaosphatase and beta-D-galactosidase are widely used as labels in the development of enzyme immunoassays (EIAs). Enzyme beta-lactamase, though introduced as a label in late seventies has not yet become very popular inspite of having the necessary features of an enzyme to be used in EIAs. The present article reviews assays developed with this enzyme, highlights its salient features and brings out an argument in favour of its wide spread use in EIAs.

Alkaline Phosphatase↗

PubMed2004

New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers.

Methane-oxidising microbial communities are studied intensively because of their importance for global methane cycling. A suite of molecular microbial techniques has been applied to the study of these communities. Denaturing gradient gel electrophoresis (DGGE) is a diversity screening tool combining high sample throughput with phylogenetic information of high resolution. The existing 16S rRNA-based DGGE assays available for methane-oxidising bacteria suffer from low-specificity, low phylogentic information due to the length of the amplified fragments and/or from lack of resolving power. In the present study we developed new combinations of existing primers and applied these on methane-oxidising microbial communities in a freshwater wetland marsh. The designed strategies comprised nested as well as direct amplification of environmental DNA. Successful application of direct amplification using combinations of universal and specific primers circumvents the nested designs currently used. All developed assays resulted in identical community profiles in wetland soil cores with Methylobacter sp. and Methylocystis sp.-related sequences. Changes in the occurrence of Methylobacter-related sequences with depth in the soil profile may be related to the decrease in methane-oxidizing activity.

DNA Primers↗

PubMed1993

Bioassay of complex mixtures of indoor air pollutants.

The development of short-term genetic bioassays has made it possible to conduct mutagenicity studies on complex mixtures of indoor air pollutants. Although most of the studies have used the standard S. typhimurium/microsome reversion assay developed by Ames et al. (1975), the development of microsuspension mutagenesis methods (see Methods 18 and 19, this volume) has facilitated wider use of bioassays in indoor air pollution studies. It is clear from the studies reviewed here that environmental tobacco smoke is the major source of mutagens indoors. Other sources which produce significant, but much smaller, increases in indoor air mutagenicity include outdoor air and other indoor combustion sources (e.g., cooking, kerosene heaters and open fireplaces burning wood). These studies have been based on measurement of the bacterial mutagenicity of extracts from particulate matter. A limited number of reports, however, indicate that volatile and semi-volatile constituents of indoor air may also be mutagenic and deserve further study.

Air Pollution, Indoor↗

PubMed1992

Accumulation of the 126 kDa protein of tobacco mosaic virus during systemic infection analysed by immunocytochemistry and ELISA.

Systemic infection of tobacco with tobacco mosaic virus (TMV) strain WU1, is accompanied by massive accumulation of the virus-coded non-structural 126 kDa protein in X-bodies. The development of X-bodies and the time course of the increase in 126 kDa protein in systemically infected leaves were analyzed by immunocytochemistry and ELISA, respectively, using an antiserum raised against a fusion protein of beta-galactosidase and part of the 126 kDa protein. The ELISA assay developed enabled routine detection of viral 126 kDa (as well as 183 kDa) protein in samples of less than 5 mg of systemically infected leaves. Plants were inoculated by differential temperature treatment, whereafter the accumulation of 126 kDa protein was related to viral multiplication, the development of X-bodies and the formation of symptoms. Both 126 kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h. By electron microscopical analysis small X-bodies, weakly labelled with antibodies against the 126 kDa protein, were detected as early as 24 h after transfer. At this stage they were not associated with nuclei. Thereafter, however, X-bodies increased in size and 126 kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves that developed mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations are consistent with the hypothesis that association of X-bodies with nuclei may lead to symptom induction, when the leaf is invaded by the virus early in its development.

Cell Compartmentation↗

PubMed1996

Radioimmunoassay of free thyroxine (T4) using 125I-labeled T4-IgG complex with very large molecular weight.

We describe a double-antibody radioimmunoassay for the determination of free thyroxine (FT4) in serum using a 125I-labeled T4-IgG conjugate of very large molecular weight (approximately 1,000,000) as tracer. The binding of this conjugate to the anti-T4 antibody was found to be satisfactory and independent of the T4-binding protein concentration. The T4 sequestration by the antibody was limited and the sample dilution used in the final assay mixture was low. The results determined by the assay developed for samples from hypo-, hyper-, euthyroid subjects and from non-thyroidal-illness patients were well correlated with those obtained by a free T4-equilibrium dialysis kit (Nichols). The detection limit of the assay was 0.41 pmol/l and the range covered by assay standard solutions was 2.4-121 pmol/l. The FT4 concentrations in serum as determined by this assay were 9.6-31 pmol/l (mean 21 pmol/l) for 46 euthyroid serum samples; 6.7-47 pmol/l (mean 23.5 pmol/l) for 27 non-thyroidal illness; and 4.2-25.8 pmol/l (mean 11.6 pmol/l) for 19 pregnant women.

Female↗

PubMed1993

A new test for infection by Entamoeba histolytica.

Entamoeba histolytica is a common parasite of the human intestine, and in a significant percentage of cases it invades the tissues. Virulent strains of the organism produce the proteinose histolysain, which almost certainly facilitates the invasive behaviour. Histolysoin has been isolated, a selective assay developed, and specific antisera raised against the enzyme. As described here by Alfredo Luaces, Lyda Osorio and Alan Barren, the ENZYMEBA test for infection by E. histolytica combines the specificities of both antibodies and enzyme assay to achieve a high degree of sensitivity and selectivity. Circulating antibodies to histolysain were detected in a solid-phase enzyme immunoassay, the results suggesting that a humoral immune response is induced by histolysain during the formation of liver abscess.

Journal Article↗