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Results for “Assay development”

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PubMed1988

Ig class and IgG subclass responses to Treponema pallidum in patients with syphilis.

The Ig class and IgG subclasses of anti-Treponema pallidum antibodies in human serum were quantified using solid-phase enzyme-linked immunosorbent assays. Development of these assays with monoclonal antibodies, each specific for a human immunoglobulin class or IgG subclass, provided quantitative data concerning the major antibody specificities. In patients with primary syphilis, anti-T. pallidum activity was limited almost exclusively to IgG1 and IgM. Coordinate, restricted expression of IgG1 and IgG3 responses in T. pallidum-specific assays was observed with sera from patients with active secondary syphilis. IgG1 and IgG3 accounted for roughly 53 and 43% of the total anti-treponemal IgG antibody activity, respectively. While IgM antibody levels were elevated in the patients with secondary syphilis, IgG2 and IgG4 levels, if present at all, represented less than 10 and 2% of the total IgG activity, respectively. Ig in sera from patients who had been treated adequately for secondary syphilis were restricted almost entirely to IgG3 and IgG1. Considering the low level of IgG3 in serum, disproportionately high percentages of antitreponemal antibodies were found in this subclass during and after treatment for secondary syphilis. The restricted, coexpression of the IgG1 and IgG3 isotypes may reflect the close genetic linkage of the gamma 1 and gamma 3 genes and possibly the impact of immunoregulatory mechanisms in response to the induction and expression of autoantibodies which arise during the course of secondary syphilis.

Adolescent↗

PubMed2006

Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.

14-3-3 Proteins↗

PubMed1992

In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes.

In vitro assays have been developed for three membrane-bound carotenogenic enzymes, phytoene desaturase, lycopene cyclase and beta-carotene hydroxylase, expressed in Escherichia coli. Transformants of E. coli containing different deletion constructs of the Erwinia herbicola carotenogenic gene cluster were employed, allowing the estimation of enzyme activities without interference from subsequent reactions. New HPLC systems were developed to separate substrates and reaction products enabling the determination of radioactivity on-line. The newly developed assays facilitate the purification of these enzymes which have never been isolated before.

Carotenoids↗

PubMed2002

Development of PCR assay for differentiation of some important wild animal meat of Sri Lanka.

A polymerase chain reaction (PCR) assay was developed to differentiate meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon hog deer (A. porcius oryzus), Ceylon sambhur (Cervus unicolor unicolor) and barking deer (Muntiacus muntijak malabaricus) from meat of cattle, goat, buffalo, pig, dog and sheep. A set of primers was designed according to the sequence of the mitochondrial cytochrome b gene of C. elaphus canadensis and by PCR amplification about 450 bp band was observed for all four animal species and these primers were not cross reacted with DNA of other animal species tested in the study under the tested reaction conditions. A band of 649 bp size was observed for all animal species when DNA was amplified with the universal primers and that indicated the presence of mitochondrial DNA in the samples. Further, the results indicated that this technique was sensitive enough to differentiate rotten meat, at least 5 days after the killing of an animal. Under these PCR conditions, the DNA of bacteria, which is involved in decomposition of meat, was not amplified with both universal and specific primers. However, the method was not sensitive enough in differentiating cooked meat of these species. Slaughtering of these four wild animal species is banned, but the animals are being killed illegally. Lack of meat identification methods has been identified as one of the major constraints to implement legal procedures and conserve biodiversity in the country.

Animals↗

PubMed2001

Galactan biosynthesis in Mycobacterium tuberculosis. Identification of a bifunctional UDP-galactofuranosyltransferase.

The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.

Bacterial Proteins↗

PubMed2003

Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding.

To quantitatively understand the binding affinity and target selectivity of small-molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub- optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.

Aminoglycosides↗

PubMed1997

Quantitative measurement of HCV RNA in the serum: a comparison of three assays based on different principles.

Quantitative measurement of hepatitis C virus (HCV) RNA is useful in patients with chronic hepatitis C, especially with interferon treatment. We examined the clinical usefulness of the AMPLICOR monitor assay, a newly developed assay for quantitative measurement, by comparing it with two other assays with different principles. A total of 48 patients with chronic hepatitis C who were treated with interferon-alpha (IFN-alpha) were studied: 19 were complete responders and 29 were non-responders. Hepatitis C virus RNA was measured quantitatively by AMPLICOR, branched DNA (bDNA) probe, and competitive polymerase chain reaction (C-PCR) assays. An internal quantification standard was used in the AMPLICOR assay. A cDNA competitor with a deletion of 15 base pairs in the middle portion was used in the C-PCR method. The concentration of HCV RNA was significantly correlated between the three assays adopted in this study. Sensitivity of assays was 100% by C-PCR, 90% by AMPLICOR and 69% by bDNA assays. The active quantitative range was best with the C-PCR assay and worst with the bDNA assay. The bDNA assay had a tendency to exhibit lower values for patients with serotype 2 than did the other two assays. The predictive rate of the long-term response to IFN-alpha therapy, before its initiation, was over 75% in all three assays. The predictive rate just after completing IFN-alpha therapy was as high as 80% by C-PCR and the AMPLICOR assays, but was low (58%) with the bDNA assay. The handling of the bDNA and AMPLICOR assays was much easier than the C-PCR assay, which required time and skill. These results indicate that the AMPLICOR assay is a simple and reliable method for measuring the serum concentrations of HCV RNA, and thus is suitable for clinical application.

Adult↗

PubMed1977

Mutagenic studies of folic acid antagonists.

Compounds that compete with folic acid (folic acid antagonists [FAAs]) become limited in their usefulness in the treatment of leukemia, malaria, and bacterial infections by the rapid development of resistance. Assays of the plasma levels of certain of these FAAs led to the observation, in about 25% of the determinations, that a higher density of growth of Streptococcus faecium var. durans (ATCC 8043) was obtained at an FAA concentration just below the completely inhibitory level than at one-half this concentration. This and other considerations suggested that FAAs may act not only as selective agents for resistant organisms but also as mutagens. Seven FAAs including amethopterin, pyrimethamine, trimethoprim, chlorguanide triazine, an experimental quinazoline, WR-158,122, and two experimental triazines, WR-99,210 and WR-38,839, were tested for mutagenicity in the Salmonella reversion assay developed by Ames et al. (1975). All were found to be negative for strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without microsomal activation. These compounds were then tested as mutagens for three traits in the folic acid-requiring S. faecium. FAAs were shown to cause mutations to folic acid independence, rifampin resistance, and FAA resistance. It is postulated that the FAAs induce mutations by causing thymine deprivation in the folic acid-requiring host.

Culture Media↗

PubMed1983

Determination of ethimizol and two of its metabolites in serum by high-pressure liquid chromatography.

A liquid chromatographic assay has been developed for the determination of ethimizol and two of its metabolites in serum. The analysed compounds are preseparated from serum by a micro-column packed with an octadecylsilanized silica gel. Components of the micro-column eluate are analysed on a silica-gel-packed column by means of a high-performance liquid chromatograph fitted with a photometric detector. At 262 nm the detection limit of the injected amount of ethimizol is about 5 ng. The mass spectra of two ethimizol metabolites isolated from rat serum are presented. The suitability of the developed assay for pharmacokinetic studies of ethimizol is demonstrated.

Animals↗

PubMed2007

Characteristics and value of directed algorithms in high content screening.

High content screening requires image processing algorithms that can accurately and robustly analyze large image numbers without requiring human intervention. Thus, a suite of algorithms that are directed by an understanding of the biology being studied was developed for the optimized automated acquisition and quantitation of cellular images. Two categories of directed algorithms were developed: Developer Tools for assay development and Specific Algorithms for turnkey screening of specific biological situations. The same basic sequence of analysis steps are used in these directed algorithms: 1. Primary object identification. 2. Measurement of primary object properties. 3. Identification and measurements of associated targets. 4. Analysis of raw measurements for specific biological problems. The detailed application of these steps is guided by the biology being studied and the expected phenotypic changes. Most cell biological problems to be analyzed using high content screening can be categorized by either the phenotype of the problem or labeling pattern, or by a standard biological response behavior of the cells. This enables application of directed algorithms optimized for these categories. Examples of the use of directed algorithms for specific categories are discussed, as well as the detailed analysis steps for a specific directed algorithm.

Algorithms↗

PubMed2001

DNA technology, the clinical laboratory, and the future.

OBJECTIVE: To review the advances in clinically useful molecular biological techniques and their applications in clinical practice as presented at the Ninth Annual William Beaumont Hospital DNA Symposium. DATA SOURCES: The 10 manuscripts submitted were reviewed and their major findings were compared with literature on the same topic. STUDY SELECTION: One manuscript reviewed the development of pharmacogenetics, 3 described analytic approaches to detect aneuploidy or cancer, 1 described transcription factor E2F-1 increase during apoptosis, 2 reported on genetic and pharmacologic factors that influence platelet aggregation, 2 described molecular methods for detecting long QT syndrome or mycobacteria, and 1 reported a modification in collection of buccal DNA. DATA SYNTHESIS: Genomic and proteomic approaches to develop clinically useful assays have been successful. Aneuploidy can be easily detected by comparative genomic hybridization, which does not require cell culture like cytogenetics. Mutations have been characterized for a variety of hereditary cancer syndromes, 2 inherited long QT syndromes, and thromboembolism. PlA1 and PlA2 polymorphisms in platelets are associated with a difference in aggregation inhibition by estrogen, another example of genotypic pharmacogenetics. Protein expression differences may define colorectal cancer stage and explain apoptotic signal transduction. Mycobacterial detection by nucleic acid amplification and simplified buccal DNA collection demonstrate cost-effective strategies. CONCLUSION: The working draft of the Human Genome Project is completed and the number of clinically useful molecular pathologic techniques and assays will expand as additional disease-associated mutations are defined. Expanded use of database software for genomic and proteomic screening should increase the efficiency of clinical useful assay development.

Biotechnology↗

PubMed1996

Delineation of immunodominant epitopes of human T-lymphotropic virus types I and II and their usefulness in developing serologic assays for detection of antibodies to HTLV-I and HTLV-II.

Several immunodominant B-cell epitopes have been mapped in the structural (gag, env) and regulatory (tax, rex) proteins of human T-lymphotropic virus type I (HTLV-I) and HTLV-II. Identification of these immunogenic epitopes not only has allowed the development of sensitive and specific serologic assays, but also has provided a tool to study correlates of host pathogenesis, viral transmission, and protection. The major immunogenic epitopes for HTLV-I are located at the C terminus of p19gag, the central and carboxyl terminus of gp46env, the central region of transmembrane envelope glycoprotein (p21e), and the carboxyl terminus of p40tax. Similarly, HTLV-II epitopes are located at the amino terminus and central region of gp46env, the central region of p21e, and the carboxyl terminus of p40tax. The transmembrane epitopes of HTLV-I and HTLV-II share extensive homologies and represent a highly cross-reactive region, while the remaining regions represent HTLV type-specific epitopes. The transmembrane epitope has been commonly used for detecting antibodies in both HTLV-I and HTLV-II-infected persons, whereas central envelope epitopes have been exploited for serologic differentiation between HTLV-I and HTLV-II. This detailed analysis at the epitope level has resulted in the development of highly sensitive and specific screening and confirmation assays for detection of antibodies to HTLVs.

Antibodies, Viral↗

PubMed1985

In vitro evaluation of anticancer drugs in relation to development of drug resistance in the human tumor clonogenic assay.

The efficacy of anticancer drugs against ovarian cancer, breast cancer, and colorectal cancer has been evaluated in vitro by the human tumor clonogenic assay developed by Hamburger and Salmon. The in vitro colony assay method used in this study is a minor modification of their method and was used in 83 patients with ovarian cancer, 47 patients with breast cancer, and 13 patients with colorectal cancer. The total numbers of assays performed in vitro were 258 for ovarian cancer, 87 for breast cancer, and 38 for colorectal cancer. The average chemosensitivity rates to single agents tested were 35% and 32% in the untreated patients with ovarian and breast cancer, respectively. In contrast to this result, the chemosensitivity rate of the untreated patients with colorectal cancer was only 16%. Consisting the clinical efficacy of anticancer drugs against these tumors, these results suggest that there is a correlation between chemosensitivity in the human tumor clonogenic assay and clinical responsiveness. In this assay the chemosensitivity in specimens from ovarian cancer patients who had had prior chemotherapy was significantly lower than in those from nonpretreated patients (P less than 0.05). This seems to indicate the development of drug resistance after treatment with anticancer drugs. These results suggest that the human tumor clonogenic assay is a useful tool for the evaluation of antitumor effects of drugs in vitro.

Antineoplastic Agents↗

PubMed2005

Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCR.

A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis.

Humans↗

PubMed1991

[Basic study on in vitro chemosensitivity tests].

By means of 3 different kinds of in vitro chemosensitivity testings--(1) a nuclear damage assay developed by us, (2) MTT assay, and (3) colony formation inhibition assay--we examined the sensitivity of 8 kinds of human ovarian cancer cell lines to various anticancer drugs. The sensitivity of in vivo xenografts of the cell lines in nude mice to anticancer drugs was also examined by inhibition of the tumor growth. The in vitro--in vivo correlation of sensitivity was studied in respect to both sensitivity and specificity rates. 1. Different active anticancer drugs were screened among the 3 in vitro chemosensitivity testings in the same human ovarian cancer cell line. 2. The in vitro--in vivo correlation of the nuclear damage assay (sensitivity 50%, specificity 94%) was the highest among the 3 testings. The nuclear damage assay which we developed therefore seemed to be the most useful assay method for clinical use.

Animals↗

PubMed1994

Developing multianalyte assays.

Multianalyte 'binding' assays represent a major advance in microanalytical technology for the measurement of substances of biological importance. Their further development should facilitate sensitive and reproducible quantification of analytes in many areas that are currently problematic, including diagnostic medicine and the standardization of biologicals.

Forecasting↗

PubMed1985

Potential uses of sea urchin embryos for identifying toxic chemicals: description of a bioassay incorporating cytologic, cytogenetic and embryologic endpoints.

A method for evaluating pollutant genotoxicity, embryotoxicity and teratogenicity using sea urchin embryos has been developed and was tested using benzo(a)pyrene (BP). Initial results suggested that the bioassay may be a sensitive indicator of pollutant toxicity and mutagenicity since several endpoints can be simultaneously assessed. The bioassay is rapid, inexpensive and appears applicable to a variety of toxicants and delivery methods. The test is based upon the standard 48 h sea urchin development assay and incorporates cytologic-cytogenetic analysis of embryos. Following toxic exposure of gametes, fertilization success is assessed. Embryos then develop for 48 h at which time survival and teratogenesis are evaluated. A subsample of embryos is stained and dissociated into monolayers and mitotic configurations are examined using light microscopy. Embryo mitotic rates are used as an indicator of overall embryonic health. Cytotoxic effects are concomitantly evaluated. Genotoxicity is measured using two methods: (1) anaphase aberration analysis, a technique which assesses abnormalities in the chromosome configurations (such as bridges and fragments) as the groups of chromosomes move to opposite poles and (2) micronucleus formation, a procedure examining the incidence of smaller, secondary nuclei composed of whole chromosomes or chromatid fragments. These two measurements preclude the need to examine individual chromosomes for deletions and exchanges, a laborious process in most aquatic organisms which possess numerous relatively small chromosomes. This genotoxicity-teratogenicity test appears promising for laboratory evaluations of individual substances or of complex chemical mixtures as well as for environmental monitoring of nearshore areas. The standard development assay has been used to screen pharmaceuticals and environmental contaminants and some recent investigations have included mitotic aberration analysis. Experiments in our laboratory suggest that the genotoxicity-teratogenicity test may be a feasible approach to field monitoring. Mutagen loads of spawning adult urchins could be assessed by conducting cytologic-cytogenetic analysis of resulting embryos although initial studies suggest that this method is less sensitive than direct embryo exposures.

Animal Testing Alternatives↗

PubMed2003

Rhesus monkey rhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication.

Rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Lack of an efficient culture system to grow high titers of virus, and the lack of an in vivo animal model system, has hampered the study of KSHV replication and pathogenesis. RRV is capable of replicating to high titers on fibroblasts, thus facilitating the construction of recombinant rhadinoviruses. In addition, the ability to experimentally infect naïve rhesus macaques with RRV makes it an excellent model system to study gamma-herpesvirus replication. Our study describes, for the first time, the construction of a GFP-expressing RRV recombinant virus using a traditional homologous recombination strategy. We have also developed two new methods for determining viral titers of RRV including a traditional viral plaque assay and a quantitative real-time PCR assay. We have compared the replication of wild-type RRV with that of the RRV-GFP recombinant virus in one-step growth curves. We have also measured the sensitivity of RRV to a small panel of antiviral drugs. The development of both the recombination strategy and the viral quantitation assays for RRV will lay the foundation for future studies to evaluate the contribution of individual genes to viral replication both in vitro and in vivo.

Animals↗