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PubMed2006

Phage display mediated immuno-PCR.

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

Capsid Proteins↗

PubMed1988

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).

Antibodies, Monoclonal↗

PubMed2005

Fluorescence in situ hybridisation analysis of chromosomal aberrations in gastric tissue: the potential involvement of Helicobacter pylori.

In this series of experiments, a novel protocol was developed whereby gastric cells were collected using endoscopic cytology brush techniques, and prepared, such that interphase fluorescence in situ hybridization (FISH) could be performed. In total, 80 distinct histological samples from 37 patients were studied using four chromosome probes (over 32,000 cells analysed). Studies have previously identified abnormalities of these four chromosomes in upper GI tumours. Using premalignant tissues, we aimed to determine how early in Correa's pathway to gastric cancer these chromosome abnormalities occurred. Aneuploidy of chromosomes 4, 8, 20 and 17(p53) was detected in histologically normal gastric mucosa, as well as in gastritis, intestinal metaplasia, dysplasia and cancer samples. The levels of aneuploidy increased as disease severity increased. Amplification of chromosome 4 and chromosome 20, and deletion of chromosome 17(p53) were the more common findings. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. Helicobacter pylori infection was determined in premalignant tissue using histological analysis and PCR technology. Detection rates were comparable. PCR was used to subtype H. pylori for CagA status. The amplification of chromosome 4 in gastric tissue was significantly more prevalent in H. pylori-positive patients (n=7) compared to H. pylori-negative patients (n=11), possibly reflecting a role for chromosome 4 amplification in H. pylori-induced gastric cancer. The more virulent CagA strain of H. pylori was associated with increased disease pathology and chromosomal abnormalities, although numbers were small (CagA+ n=3, CagA- n=4). Finally, in vitro work demonstrated that the aneuploidy induced in a human cell line after exposure to the reactive oxygen species (ROS) hydrogen peroxide was similar to that already shown in the gastric cancer pathway, and may further strengthen the hypothesis that H. pylori causes gastric cancer progression via an ROS-mediated mechanism.

Aged↗

PubMed2004

Emerging tick-borne infections: rediscovered and better characterized, or truly 'new' ?

The emergence of Lyme borreliosis as a public health burden within the last two decades has stimulated renewed interest in tick-borne infections. This attention towards ticks, coupled with advances in detection technologies, has promoted the recognition of diverse emergent or potentially emerging infections, such as monocytic and granulocytic ehrlichiosis, local variants of spotted fever group rickettsioses, WA-1 babesiosis, or a Lyme disease mimic (Masters' Disease). The distribution of pathogens associated with well-described tick-borne zoonoses such as human babesiosis due to Babesia microti or B. divergens seems wider than previously thought. Bartonellae, previously known to be maintained by fleas, lice or sandflies, have been detected within ticks. Purported 'new' agents, mainly identified by sequencing of PCR products and comparison with those sequences present in GenBank, are being increasingly reported from ticks. We briefly review the diversity of these infectious agents, identify aetiological enigmas that remain to be solved, and provide a reminder about 'old friends' that should not be forgotten in our pursuit of novelty. We suggest that newly recognised agents or tick/pathogen associations receive careful scrutiny before being declared as potential public health burdens.

Animals↗

PubMedDate not supplied

Monitoring of RNA clearance in a novel plasmid DNA purification process.

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.

Biochemistry↗

PubMed1997

Recent developments in the epidemiology of prostate cancer.

The purpose of this review is to examine the implications of recent trends in prostate cancer incidence and mortality and to consider recent progress of epidemiological research on this disease. The incidence and mortality rates for prostate cancer are changing throughout the world at an accelerating pace. Much of the increase in incidence is related to changes in detection technology, but increases in mortality rates suggest other factors are involved. Recent research has focused on race-related difference, diet and related lifestyle exposures, the aetiologic significance of vasectomy and, patterns of familial aggregation. Continued monitoring of trends in incidence and mortality will be important as prostate cancer becomes a greater public health burden worldwide. Epidemiological research has identified several possible risk factors which may be useful for prostate cancer prevention and targeting high-risk individuals for early detection intervention. Additional research may confirm and refine understanding of prostate cancer aetiology.

Diet↗

PubMed2006

Microelectrode array studies of basal and potassium-evoked release of L-glutamate in the anesthetized rat brain.

L-glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system. It plays major roles in normal neurophysiology and many brain disorders by binding to membrane-bound Glu receptors. To overcome the spatial and temporal limitations encountered in previous in vivo extracellular Glu studies, we employed enzyme-coated microelectrode arrays to measure both basal and potassium-evoked release of Glu in the anesthetized rat brain. We also addressed the question of signal identity, which is the predominant criticism of these recording technologies. In vivo self-referencing recordings demonstrated that our Glu signals were both enzyme- and voltage-dependent, supporting the identity of L-glutamate. In addition, basal Glu was actively regulated, tetrodotoxin (TTX)-dependent, and measured in the low micromolar range (approximately 2 microm) using multiple self-referencing subtraction approaches for identification of Glu. Moreover, potassium-evoked Glu release exhibited fast kinetics that were concentration-dependent and reproducible. These data support the hypothesis that Glu release is highly regulated, requiring detection technologies that must be very close to the synapse and measure on a second-by-second basis to best characterize the dynamics of the Glu system.

Anesthetics↗

PubMed2005

Application of Affymetrix array and Massively Parallel Signature Sequencing for identification of genes involved in prostate cancer progression.

BACKGROUND: Affymetrix GeneChip Array and Massively Parallel Signature Sequencing (MPSS) are two high throughput methodologies used to profile transcriptomes. Each method has certain strengths and weaknesses; however, no comparison has been made between the data derived from Affymetrix arrays and MPSS. In this study, two lineage-related prostate cancer cell lines, LNCaP and C4-2, were used for transcriptome analysis with the aim of identifying genes associated with prostate cancer progression. METHODS: Affymetrix GeneChip array and MPSS analyses were performed. Data was analyzed with GeneSpring 6.2 and in-house perl scripts. Expression array results were verified with RT-PCR. RESULTS: Comparison of the data revealed that both technologies detected genes the other did not. In LNCaP, 3,180 genes were only detected by Affymetrix and 1,169 genes were only detected by MPSS. Similarly, in C4-2, 4,121 genes were only detected by Affymetrix and 1,014 genes were only detected by MPSS. Analysis of the combined transcriptomes identified 66 genes unique to LNCaP cells and 33 genes unique to C4-2 cells. Expression analysis of these genes in prostate cancer specimens showed CA1 to be highly expressed in bone metastasis but not expressed in primary tumor and EPHA7 to be expressed in normal prostate and primary tumor but not bone metastasis. CONCLUSION: Our data indicates that transcriptome profiling with a single methodology will not fully assess the expression of all genes in a cell line. A combination of transcription profiling technologies such as DNA array and MPSS provides a more robust means to assess the expression profile of an RNA sample. Finally, genes that were differentially expressed in cell lines were also differentially expressed in primary prostate cancer and its metastases.

Cell Line, Tumor↗

PubMed2006

Surface plasmon resonance-enabled mass spectrometry arrays.

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.

Antibodies↗

PubMed2001

A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction.

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogeneous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology.

Animals↗

PubMed2004

Identifying biomarkers and surrogates of tumors (cancer biometrics): correlation with immunotherapies and immune cells.

The presence of inflammatory cells within cancer has been described for quite some time by pathologists, with generally improved outcome associated with their presence in various epithelial neoplasms. Most remarkably, this has included dendritic cells and T cells but more recently NK cells as well. Coupled with the rapid evolution of molecular technology, microarray analyses of primary tumors, serum and tumor proteomics, tumor capture analyses in the peripheral blood (together with quantitative RT-PCR), and novel histochemical markers and tissue microarrays, this provides the opportunity to establish a more effective means to study and classify into subsets various forms of cancer. Much of the current controversy in cancer diagnosis and pathologic assessment of prognosis lies in the application of these techniques in concert with other molecular tools including DNA microarrays, expression of histochemically defined cytokines, proangiogenic factors, and oncogene products, and correlating this with clinical relevance. Molecular detection technologies such as reverse transcriptase polymerase chain reaction, proteomics, and microarray analyses will be validated based on their integration with conventional cancer pathology and cancer diagnostics. Further work is needed to establish which cancer biomarkers and surrogates should be routinely measured and in which settings, and determining the appropriate sample size for such assays that can be validated in retrospective and prospective clinical studies. The ability to integrate these rapidly evolving strategies will consume much of our coordinate effort in cancer and cancer therapeutics for the near future.

Biomarkers, Tumor↗

PubMed2006

Technical performance of the Luxel Al(2)O(3):C optically stimulated luminescence dosemeter element at radiation oncology and nuclear accident dose levels.

The dose ranges typical for radiation oncology and nuclear accident dosimetry are on the order of 2-70 Gy and 0.1-5 Gy, respectively. In terms of solid-state passive dosimetry, thermoluminescent (TL) materials historically have been used extensively for these two applications, with silver-halide, leuco-dye and BaFBr:Eu-based films being used on a more limited basis than TL for radiation oncology. This present work provides results on the performance of a film based on an aluminum oxide, Al(2)O(3):C, for these dosimetry applications, using the optically stimulated luminescence (OSL) readout method. There have been few investigations of Al(2)O(3):C performance at radiation oncology and nuclear accident dose levels, and these have included minimal dosimetric and environmental effects information. Based on investigations already published, the authors of this present study determined that overall improvements over film and TLDs for this Al(2)O(3):C OSL technology at radiation oncology and nuclear accident dose levels may include (1) a more tissue-equivalent response to photons compared to X-ray film, (2) higher sensitivity, (3) ability to reread dosemeters and (4) diagnostic capability using small-area imaging. The results of the present investigation indicate that additional favourable performance characteristics for the Al(2)O(3):C dosemeter are a wide dynamic range (0.001-100 Gy), a response insensitive to temperature and moisture over a wide range, negligible dose rate dependence, and minimal change in post-irradiation response. As a radiation detection medium, this OSL phosphor offers an assortment of dosimetry properties that will permit it to compete with current radiation detection technologies such as silver-halide, leuco-dye and photostimulable-phosphor-based films, as well as TLDs.

Aluminum Oxide↗

PubMed2002

Stains, labels and detection strategies for nucleic acids assays.

Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the final decade of the 20th century (1991-2000). During this period, important families of cyanine dyes were developed for sensitive detection of double-stranded DNA, single-stranded DNA, and oligo(deoxy)nucleotides in gels and in solution, and families of energy transfer primers were produced for DNA sequencing applications. The continuing quest for improved labels for hybridization assays has produced a series of candidate labels including genes encoding enzymes, microparticles (e.g. quantum dots, nanocrystals, phosphors), and new examples of the fluorophore (e.g. cyanine dyes) and enzyme class of labels (e.g. firefly luciferase mutants). Label detection technologies for use in northern and southern blotting assays have focused on luminescent methods, particularly enhanced chemiluminescence for peroxidase labels and adamantyl 1,2-dioxetanes for alkaline phosphatase labels. Sets of labels have been selected to meet the demands of multicolour assays (e.g. four-colour sequencing and single nucleotide primer extension assays). Non-separation assay formats have emerged based on fluorescence polarization, fluorescence energy transfer (TaqMan, molecular beacons) and channelling principles. Microanalytical devices (microchips), high-throughput simultaneous test arrays (microarrays, gene chips), capillary electrophoretic analysis and dipstick devices have presented new challenges and requirements for nucleic acid detection, and fluorescent methods currently dominate in many of these applications.

Alkaline Phosphatase↗

PubMed2004

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis.

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band.

Antibodies↗

PubMed2001

Assessing uterine receptivity in 2001: ultrasonographic glances at the new millennium.

The understanding and control of embryo implantation represents the major challenge for assisted reproductive technologies. Along with developments in basic research and efforts to optimize embryo quality, the improvement of noninvasive and reliable methods to assess uterine receptivity constitutes an important step toward meeting such a challenge. Today, ultrasound-based approaches to evaluate endometrial echogenicity and uterine perfusion and contractility are available for practical use. Increasing evidence indicates that echogenic patterns of the endometrium reflect histologic processes that are involved in the establishment of receptivity. This constitutes a possible explanation for the reported association between premature hyperechogenic patterns of the endometrium and poor implantation rates. Nevertheless, additional studies aiming at correlating further morpho-biochemical events in the endometrium with its echogenicity patterns are needed. Further, developments in vascular assessment by Doppler, Doppler-related, and vascular detection technologies will also be instrumental in monitoring and improving vascular changes that lead to uterine receptivity. Finally, data supporting the hypothesis that uterine contractility, as visualized by ultrasound, influences in vitro fertilization-embryo transfer (IVF-ET) pregnancy rates encourage further investigation on both the regulation and control of uterine contractions. This article discusses some of the advantages and limitations of ultrasonographic assessments of uterine receptivity in the perspective of the new millennium.

Embryo Implantation↗

PubMed2003

Cationic comb-type copolymers for DNA analysis.

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

Terahertz circular dichroism spectroscopy: a potential approach to the in situ detection of life's metabolic and genetic machinery.

We propose a terahertz (far-infrared) circular dichroism-based life-detection technology that may provide a universal and unequivocal spectroscopic signature of living systems regardless of their genesis. We argue that, irrespective of the specifics of their chemistry, all life forms will employ well-structured, chiral, stereochemically pure macromolecules (>500 atoms) as the catalysts with which they perform their metabolic and replicative functions. We also argue that nearly all such macromolecules will absorb strongly at terahertz frequencies and exhibit significant circular dichroism, and that this circular dichroism unambiguously distinguishes biological from abiological materials. Lastly, we describe several approaches to the fabrication of a terahertz circular dichroism spectrometer and provide preliminary experimental indications of their feasibility. Because terahertz circular dichroism signals arise from the molecular machinery necessary to carry out life's metabolic and genetic processes, this life-detection method differs fundamentally from more well-established approaches based on the detection of isotopic fractionation, "signature" carbon compounds, disequilibria, or other by-products of metabolism. Moreover, terahertz circular dichroism spectroscopy detects this machinery in a manner that makes few, if any, assumptions as to its chemical nature or the processes that it performs.

Circular Dichroism↗

PubMed2006

Analysis of sulphonamide residues in edible animal products: a review.

The methods of analysis for sulphonamide residues in edible animal products are reviewed. Sulphonamides are widely used for therapeutic and prophylactic purposes in both humans and animals, sometimes as growth promoters as additives in animal feed. As a result of their widespread use, there is concern about whether the levels used of these drugs can generate serious problems in human health, e.g., allergic or toxic reactions. Several methods for the determination of sulphonamides have been reported in the literature and this review considers high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), gas chromatography (GC), thin-layer chromatography (TLC), high-performance capillary electrophoresis (HPCE), enzyme-linked immunosorbant assay (ELISA), biosensor immunoassay (BIA) and microbiological methods. Specific aspects of analysing sulphonamides, such as sample handling, chromatographic conditions and detection methods are discussed. Methods for drug residue monitoring should be accurate, simple, economical in both time and cost, and capable of detecting residues below the maximum residue limits (MRL). The current sulphonamide detection technologies are based on chromatographic methods or bacteriological growth inhibition. The instrumental methods such as HPLC and GC are both sensitive and specific, but are laborious and expensive. Because of the labour-intensive processes, only a few cases of GC methods applied to residue analysis have been published. These methods are suitable for confirmation but not for screening of large numbers of samples. Microbiological methods do not require highly specialized and expensive equipment. They also use highly homogeneous cell populations for testing and thus result in better assay precision. Although HPCE has powerful separation ability, the precision is poor and the instrument still needs to be improved. To date, this technique has not been widely applied to routine analysis. Currently, TLC has been almost replaced by other instrumental analysis. A rapid, sensitive and specific assay is required to detect positive samples in routine analysis, which can then be confirmed for the presence of sulphonamides by HPLC. Immunochemical methods such as ELISA can be simple, rapid and cost-effective, with enough sensitivity and specificity to detect small molecules. This review can be considered as a basis for further research aimed at identifying the most efficient approaches.

Animals↗