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PubMed2000

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Bacterial Proteins↗

PubMed1999

Methods of screening combinatorial libraries using immobilized or restrained receptors.

The screening of combinatorial libraries for compounds with high affinity toward drug receptors is currently a major center of attention. We describe methods recently developed for library screening that involve "constrained" receptors (either immobilized onto a surface or restrained to a compartment by some physical means). These include affinity selection chromatography, ultrafiltration assays, the scintillation proximity assay, a variety of interfacial optical techniques (surface plasmon resonance and its relatives, among others), the quartz crystal microbalance, the jet ring cell, and new interferometric assays using porous silicon to immobilize the receptor. We note some trends in assay development involving assays of membrane-bound complexes, and the coupling of two analytical methods to expand the assay resolution.

Chemistry, Pharmaceutical↗

PubMed1986

Investigation of IgG4 levels in atopic patients using a competitive inhibition assay employing biotinylated IgG4 myeloma and avidin peroxidase.

Modification of a 'sandwich' ELISA assay developed for the determination of serum IgE levels proved to be unsatisfactory for the measurement of IgG4. This was attributed to the limited capacity of the microtitre plate solid phase which required high serum dilutions in order to measure IgG4 levels. To overcome this problem a competitive inhibition assay was developed with monoclonal anti-IgG4 attached to the plate. In this system biotinylated IgG4 myeloma and sample IgG4 compete for the limited antibody binding sites present on the solid phase. The attached biotinylated myeloma is detected by addition of avidin conjugated with peroxidase and following development with substrate, IgG4 levels are calculated by reference to a calibrated inhibition curve. The inhibition ELISA assay has been used clinically to measure IgG4 levels in atopic and normal individuals and the values obtained correlated closely (r = 0.99) with the IgG4 levels determined by radial immunodiffusion. For 43 atopic dermatitis patients investigated the median IgG4 level was 1.1 g/l which was significantly elevated when compared to a median of 0.385 g/l for 60 blood donors (P less than 0.0001, Mann-Whitney U). Among the 47 hay fever patients investigated the median was 0.6 g/l which, although lower than in atopic dermatitis, was again significantly increased (P less than 0.025). Within this latter group, 25 patients were investigated for the effects of desensitization with commercial grass pollen injections. The total IgG4 showed a variable but significant rise between the start and finish of treatment (P less than 0.01 Wilcoxon signed ranks test).

Binding, Competitive↗

PubMed1993

Evaluation of colorectal cancer-associated mucin CA M43 assay in serum.

The technical performance of a newly developed assay for CA M43, a serum marker for colorectal cancer, was evaluated and its preliminary clinical potential assessed. The heterologous double-determinant enzyme immunoassay for the detection of the tumor-associated mucin CA M43 utilizes two monoclonal antibodies (CT 43 and CT 66) selected for their binding capacity to two distinct epitopes present on mucins in the sera of patients with colorectal cancer. CT 66 recognizes both Lewis(a) and sialylated Lewis(a) antigen; CT 43 is directed toward a mucin epitope of an as-yet uncharacterized structure. Precision experiments revealed interassay CVs of 11.8%, 5.9%, and 4.9% at 9.3, 11.9, and 78.9 units/mL, respectively; intraassay precision was 2.0% at 95.1 units/mL. The upper normal value was set at 7.5 units/mL, which included 99% of the values found in healthy controls. In colorectal cancer patients, CA M43 showed a positivity rate equivalent to that of carcinoembryonic antigen (CEA) and superior to that of CA 19.9, with only one CA 19.9-positive serum being negative for CA M43. Interestingly, CA M43 appeared to be complementary to CEA, with CA M43 and CEA together reaching 87% positivity in metastatic disease.

Adult↗

PubMed2004

Effect of methoxychlor on various life stages of Xenopus laevis.

The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at </=1.0 mg/l methoxychlor (maximum soluble concentration). The 30-day hind limb development studies indicated methoxychlor exposure >/=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the frequency of amplexus, fertilization, and embryo viability was also affected by adult female methoxychlor exposure, and to a lesser extent by male exposure. Overall, these results suggested that sensitivity to methoxychlor is most dramatic during the reproductive and metamorphic phases of the life cycle and least sensitive during early embryo-larval development.

Animals↗

PubMed1993

Evaluation and improvement of an enzyme-linked immunosorbent assay for the detection of isometamidium in bovine serum.

The control of bovine trypanosomiasis in Africa continues to rely heavily on the chemoprophylactic drug isometamidium (ISMM) chloride. However, despite many years of use, no methods are available that are sufficiently sensitive to measure drug levels in treated cattle. An enzyme-linked immunosorbent assay (ELISA) for the detection of ISMM in the serum of treated cattle has been developed and evaluated. Liquid-phase ISMM (sample) competes with solid-phase bound ISMM-protein conjugate for biotinylated sheep anti-ISMM IgG. The specific IgG is detected by streptavidin-peroxidase, using tetramethylbenzidine for colour development. Assay calibration is by four-parameter logistic curve-fitting. Factors contributing to absorbance variance were considered in assay optimization and improvement of precision and the lower limit of detection (approximately 0.1 ng/ml in serum). The ELISA was shown to detect serum ISMM for several months after treatment of cattle in a trypanosomiasis endemic country. The potential uses of this assay include the development of rational prophylactic drug regimens, and the indirect detection of drug-resistant trypanosomes.

Animals↗

PubMed2004

Development and characterization of liposomal disodium ascorbyl phytostanyl phosphates (FM-VP4).

The specific objectives of this project were (1) to develop liposomal disodium ascorbyl phytostanyl phosphate (FM-VP4) formulations, (2) to develop a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) assay for quantification of FM-VP4 in liposomal formulations and plasma sample, and (3) to characterize liposomal FM-VP4 formulations by finding optimal drug-to-lipid ratios and determining the degradation of FM-VP4 in liposomes. Section 2 describes an LC/MS/MS assay developed for the identification and quantification of FM-VP4 in liposomal formulations to provide estimates of drug concentrations and encapsulation efficiency. The extra step of removing plasma proteins prior to LC/MS/MS assay yields an analysis of FM-VP4 in plasma samples. Section 3 describes experiments designed to find the optimal drug-to-lipid ratio for liposomal FM-VP4 formulations by comparing encapsulation efficiencies and varying the lipid compositions. Additionally, this section details our degradation studies to determine if liposomes have any protective effects on FM-VP4; these studies tested various lipid compositions at 37 degrees C in rabbit plasma. The mechanism of how FM-VP4 lowers low-density lipoprotein (LDL) cholesterol and total cholesterol levels in various animal models is presently unknown. However, before the mechanism of action could be studied, FM-VP4 first had to be delivered efficiently into plasma or cultured cell. The low systemic bioavailability and cellular uptake of FM-VP4 further suggested the importance of finding an efficient delivery vehicle for this drug. This project proposed a framework for such delivery and paves the way for further investigation into how FM-VP4 works in vivo and in vitro.

Animals↗

PubMed2005

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

Biological Assay↗

PubMed2001

Development of a simplified assay for the detection of neutralizing antibodies to Japanese encephalitis virus.

Japanese encephalitis virus is a common cause of viral encephalitis in Asia with an estimated 45,000 cases annually. It causes significant morbidity and mortality. It is transmitted primarily by Culex mosquitoes between birds and animals, while man is thought to be an accidental, dead-end host. Since dengue is also prevalent usually in Japanese encephalitis-endemic areas, all Japanese encephalitis positive sera must be confirmed by detecting Japanese encephalitis specific neutralizing antibodies. The plaque reduction neutralization test is the gold standard for detecting and quantifying Japanese encephalitis neutralizing antibodies. This test, however, takes about a week and is carried out in 6 or 24-well plates, which limits its usage for large-scale screening. A simplified assay was developed for the detection and quantification of Japanese encephalitis neutralizing antibodies. The assay, which is carried out in 96-well plates, would be suitable for use in the mass screening of the population's immunity level as well as for use in vaccine efficacy studies.

Animals↗

PubMed1995

Analytical evaluation of a newly developed ELISA mass assay for pancreatic amylase.

OBJECTIVE: To evaluate a newly developed ELISA mass assay for Pancreatic Amylase. METHODS: The serum levels of pancreatic amylase were measured by an in-house developed ELISA assay on microtitre plate and compared with two activity assays: immunoinhibition and electrophoresis. RESULTS: The proposed method was accurate and precise, as indicated by a recovery of 102% and coefficient of variation of less than 5% for within-run and less than 10% for between-run assay. The proposed assay showed good correlation with the activity assays (r = 0.98). The mass concentrations were three times higher than the activities. The relative values (mass or activity units/upper reference limits), however, were concordant in controls and in pancreatic and non-pancreatic conditions. CONCLUSION: Our results indicate that the activity measurements and mass concentrations of pancreatic amylase in serum are comparable and interchangeable after adjusting for the reference range.

Amylases↗

PubMed2003

Comparison of ELISA and RT-PCR versus immune electron microscopy for detection of bovine torovirus (Breda virus) in calf fecal specimens.

Bovine Torovirus (BoTV) is an uncultivable enteric pathogen of cattle. Its failure to grow in vitro limits epidemiological studies, characterization of the virus, and development of diagnostic techniques. The objectives of this study were to develop and standardize an antigen-capture enzyme-linked immunosorbent assay (ELISA) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of BoTV in fecal specimens. These assays were compared with immunoelectron microscopy (IEM) to evaluate their sensitivity, specificity, and efficiency as well as their advantages and limitations. Additionally, several methods to calculate ELISA cutoff values were used and compared using a statistical approach to obtain the optimal cutoff value for the ELISA. A plate cutoff ELISA value was determined to be the best method to calculate the cutoff value. The ELISA and RT-PCR assays developed in this study identified BoTV antigen and viral nucleic acids in feces without cross-reactions with the other calf enteric viruses examined. Both assays showed good agreement with IEM, with a Kappa value of 0.86 for ELISA and 0.85 for RT-PCR. The latter exhibited the higher analytical sensitivity. On the basis of the results obtained in this study, it is recommended that no single test should be used alone in an epidemiological survey because of the observed limitations of each assay. The fast and inexpensive ELISA combined with the highly specific and sensitive RT-PCR are a practical approach for future epidemiological studies of BoTV. These results should provide other researchers with the information needed to develop similar diagnostic assays for the study of BoTV.

Animals↗

PubMed1986

A novel yeast histone deacetylase: partial characterization and development of an activity assay.

We have characterized a histone deacetylase activity associated with yeast nuclei. An unusual feature of the deacetylase is that it is not inhibited by the short-chain fatty acids n-butyrate and propionate. These short-chain fatty acids are typically potent inhibitors of histone deacetylases in eukaryotic systems. The deacetylase(s) were detected by monitoring the levels of acetylation of yeast histones during incubation of isolated yeast nuclei. The activity was optimal at 37 degrees C and at 0.1 M NaCl. The enzyme did not require divalent cations and was inhibited by Zn2+ and Cu2+. A simple activity assay was developed using as substrate, [3H]acetate-labeled histone in chicken erythrocyte nuclei. This assay was used to demonstrate that the deacetylase(s) can be extracted from yeast nuclei with 0.5 M NaCl. A gel electrophoretic analysis of the deacetylated chicken histones verified that the solubilization of incorporated radiolabel was a result of histone deacetylation, not an artifact of histone degradation by yeast proteinases.

Amidohydrolases↗

PubMed1983

Detection of unique antigenic determinants on human plasma low density lipoprotein and on delipidated apolipoprotein B.

To obtain detailed information on the role played by apolipoprotein B (apo B) in determining the structural and functional properties of human plasma low density lipoprotein, we have initiated immunochemical studies of the polypeptide. We report here the establishment of six hybridoma lines that secrete monoclonal antibodies to low density lipoprotein. In addition to recognizing antigenic determinants on low density lipoprotein, all six monoclonal antibodies react with epitope(s) on very low density, but not high density, lipoproteins. The immunoreactivity of these antibodies with low density lipoprotein and with detergent-delipidated apo B was compared in an enzyme-linked immunosorbent assay. Although all six of the antibodies reacted with the apoprotein when it was prepared in a nonionic detergent known to maintain the secondary structure of the protein, three of the six antibodies showed partial or total loss of activity with NaDodSO4- delipidated apo B. The specificity of these antibodies was tested by the ability of affinity-purified biotinylated antibodies to compete with unlabeled antibodies for antigenic sites on low density lipoprotein in a competition enzyme-linked immunosorbent assay developed with avidin-peroxidase. This competition assay allowed us to divide the antibodies into a minimum of two groups (I and II) based on the antigenic determinants on apo B that they recognized. The epitope on apo B recognized by group II antibodies was perturbed in NaDodSO4, whereas the determinant(s) on the protein reactive with group I antibodies was unaffected.

Animals↗

PubMed2005

Adding sirolimus to tacrolimus-based immunosuppression in pediatric renal transplant recipients reduces tacrolimus exposure.

In adult renal recipients, coadministration of tacrolimus (TAC) and sirolimus (SIR) results in reduced exposure to TAC at SIR doses of 2 mg/day. Eight pediatric renal recipients (median age at transplant 2.0 years, range: 1.2-12.9 years) were converted to TAC- and SIR-based immunosuppression as a rescue therapy. All patients had biopsy-proven chronic allograft nephropathy. TAC levels were measured using a commercially available EMIT assay and SIR levels with a newly developed assay based on the LC-MS MS technology. SIR was started at 0.13+/-0.05 mg/kg/day (3.51+/-1.26 mg/m2/day) in two divided doses. TAC was given at 0.14+/-0.09 mg/kg/day, resulting in a trough level of 6.3+/-2.5 ng/mL. After the addition of SIR, the median dose required to keep TAC blood trough concentrations within the target range increased by 71.2% (range: 21.9-245.4%), dose-normalized TAC exposure (AUC) decreased to 67.1% and the dose-normalized C(max), a surrogate for absorption rate, to 53.8% (both geometric means) while terminal half-life (t1/2), a pharmacokinetic parameter characterizing systemic elimination, remained unchanged (p<0.93). Adding SIR to TAC-based immunosuppression in young pediatric renal transplant recipients results in a significant decrease of TAC exposure. TAC trough levels should be monitored frequently.

Adolescent↗

PubMed2000

Real-time quantitative PCR for human herpesvirus 6 DNA.

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10(6) viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.

DNA, Viral↗

PubMed2003

Optimization of a higher throughput microsomal stability screening assay for profiling drug discovery candidates.

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.

Animals↗

PubMed1993

Variation in results of cost-effective EMIT assays on the Cobas-Bio analyser.

Following suggestions that spurious results occur with EMIT assays--so-called 'fliers'--many laboratories perform such assays in duplicate. In this study, using a Cobas-Bio centrifugal analyser and cost-effective assays developed in the authors' laboratory, complete EMIT assays were run, each with its own calibration curve, on three anti-epileptic drugs using plasma samples, and five drugs of abuse using urine samples. No serious spurious results were obtained. It is suggested that there is no inherent tendency for EMIT assays to produce 'fliers' and that when these occur they may be explained by factors common to many other types of assay.

Autoanalysis↗

PubMed1981

Milk-specific antibody measurement by elisa: development of an assay.

This paper describes the development of a simple, sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the measurement of serum IgG, IgA and IgM antibodies to cow's milk protein. The specificity of this assay was determined by preparing immunosorbents of cow's milk and human serum albumin (HSA). Fractionation of the antiserum-enzyme label by gel filtration and affinity chromatography showed that the conjugate preparation contained moieties of antiserum-enzyme, and antiserum-enzyme polymers. The preparation also contained enzyme-enzyme and antiserum-antiserum conjugates. Purification of the labeled reagent did not improve the sensitivity. The reagents and conditions used have been analyzed for purposes of standardisation.

Adsorption↗