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PubMed1995

A new luminometer for sensitive quantification by chemiluminescence of specific proteins in microtitre plates and on blot membranes.

Generally applicable technologies are described, which depend on the use of chemiluminescence and a new type of a versatile luminometer, that can measure also weak light in microtitre plates (microplates) and on blotting membranes that is especially useful for dot blot immunoassay. Applications are described using alkaline phosphatase conjugated antibodies against IgG and IgE and the reagent Lumi-Phos 530. This chemiluminescence offers advantages over the use of radioactive isotopes, densitometry and light reflection measurement on membranes and also ELISA, for sensitive quantification of e.g. specific proteins. Special procedures are described for the first time that with the mentioned reagent in agarose gel allows specific and very sensitive quantification of proteins on dot blot membranes. The luminometer, which has temperature control, is very sensitive, precise and allows efficient protocols for various assays. It thus fulfils many of the requirements for good quantification, is time-saving and in addition brings significant improvements due to very low detection limits and large linear concentration ranges that can be measured with excellent regression coefficients (r2 often about 0.999).

Alkaline Phosphatase↗
PubMedDate not supplied

A practical enzyme immunoassay for anti-acetylcholine receptor antibodies in myasthenia gravis.

A practical enzyme immunoassay (EIA) has been developed for the measurement of anti-acetylcholine receptor (AChR) antibodies in sera from patients with myasthenia gravis (MG). This system is based on the double antibody technique, using denervated rat muscle AChR labeled with horseradish peroxidase-linked alpha-bungarotoxin (HRP-alpha BGT). This method has the following advantages compared to conventional radioimmunoassay (RIA): (1) HRP-alpha BGT is more stable than [125I]alpha BGT and can be used for at least one year without any loss of the binding activity to AChR and enzymatic activity, (2) the procedure avoids the use of radioactive isotopes, and (3) the equipment for our EIA is more economical than that for RIA.

Adolescent↗
PubMed1980

Status and significance of testing for hepatitis B surface antigen and surface antibody.

Following Blumberg's discovery of hepatitis B surface antigen (HBsAg), many attempts have been made to develop several in vitro diagnostic techniques for the detection of this antigen and its homologous antibody. The two-dimensional micro-Ouchterlony immunodiffusion has been the first technique used, rapidly replaced by procedures of increasing sensitivity characterized as second-generation and the currently available third-phase tests which include radioimmunoassay (RIA), reverse passive haemagglutination (RPHA), reverse passive latex agglutination (RPLA) and enzyme immunoassay (EIA). Among these, RIA appears to be the most sensitive and specific, whereas EIA, RPHA and RPLA have the advantage of long shelf-life of stable reagents, no need for sophisticated and expensive equipment and no hazard associated with the handling of radioactive isotopes. Moreover, the sensitivity of EIA should increase by objective reading with a colorimeter. The most sensitive method for the detection of surface antibody (anti-HBs) is again RIA, whereas passive haemagglutination (PHA) had the advantage of providing titres. Finally EIA, based on inhibition of a known amount of HBsAg, has at least the same sensitivity as PHA, but has the advantage that reagents are more stable and that it permits screening for both HBsAg and anti-HBs with the same reagents at the same time. The application of these highly sensitive techniques for the detection of HBsAg and anti-HBs has resulted in a consistent reduction in the incidence of post-transfusion hepatitis type B and in a better understanding of the aetiology, epidemiology and natural history of this infection.

Antibodies, Viral↗
PubMed1987

ADPribosylation of nuclear proteins labeled with [3H]adenosine: changes during the HeLa cycle.

Cell cycle variations in the modification of histones and nonhistones by ADPribosylation were investigated. Proteins of HeLa interphase nuclei and metaphase chromosomes were radioactively labeled in vivo with [3H]adenosine. Histones of metaphase chromosomes were extensively modified by ADPribosylation, with H2B, H2A and H4 being predominant acceptors of [3H]adenosine label. For histones of interphase nuclei from synchronized cells, the highest level of 3H labeling was observed by two-dimensional gel electrophoresis to occur in S phase. The minimum level was noted in G1 phase. ADPribosylation of histones is, however, significant during all phases of the cell cycle. These conclusions were confirmed by experiments using [32P]NAD. The results with the specific inhibitor of ADPribosylation, 3-aminobenzamide, and with snake venom phosphodiesterase indicated that the radioactive isotopes were incorporated as ADPribose. Two-dimensional gels of HeLa nonhistones labeled with [3H]adenosine showed strikingly different patterns for interphase and metaphase samples. Over 100 ADPribosylated species were found for interphase nuclei, but poly(ADPribose) polymerase was the only major acceptor for metaphase chromosomes. A simple pattern was also revealed for nuclear scaffolds, with the 'lamins' and poly(ADPribose) polymerase being identifiable as modified species.

Adenosine↗
PubMed1993

The occurrence and bioavailability of radioactive 137Cs in small forest lakes in Southern Finland.

Following the Chernobyl accident in late April 1986, central Finland was subjected to considerable radioactive fallout. The radioactive isotope of caesium, (137)Cs, was potentially the most harmful isotope in the fallout because of its long half-life (30 years). (137)Cs activities remained unexpectedly low within the biota (algae, zooplankton, aquatic moss, fish) of some humic forest lakes in southern Finland compared to the clear water lakes within the same district. This observation suggested that humic substances, by binding (137)Cs chemically, may reduce its bioavailability in lake water. Our laboratory experiments (throughflow systems; gel chromatography) demonstrated that binding of (137)Cs by humic substances was negligible in untreated lake waters; only when most of the alkali metals had been removed with an ion exchange resin was any binding of (137)Cs by low molecular humic fractions apparent. Instead, the concentrations of cations (especially potassium) were of overwhelming importance for the bioavailability of (137)Cs within the lacustrine ecosystems. The concentrations of (137)Cs within food chains are expected to stay at a high level for many years especially in lakes with long water renewal times.

Journal Article↗
PubMed1986

Early detection of extravascular lung water in an inhalation injury animal model.

A rabbit inhalation injury model using a dual tracer radioactive isotope technique (Rowland et al., 1986), has been utilized with small- and medium-sized molecular weight tracers. There is oedema formation at 2 h after smoke inhalation or fluid resuscitation and especially with the combination of inhalation injury and fluid resuscitation [corrected]. This oedema appears to have decreased by 24 h post-injury. There does not appear to be any difference between the small- and medium-sized tracers in the transit times of tracer in the lung vascular compartment. The increases in extravascular water volume have been confirmed by gravimetric analyses of the lungs. Thus the pulmonary oedema in the rabbit animal model is detectable within 2 h post-injury, with a return towards normal values some time within 24 h if fluid challenged for short periods. Fluid resuscitation exacerbates the amount of oedema that developed. The onset of pulmonary oedema may therefore be earlier than previously seen clinically, and fluid resuscitation modifications may be of benefit in smoke inhalation injury.

Animals↗
PubMed1988

Diagnostic notes--lumbar BMC reproducibility as evaluated by means of dual photon absorptiometry.

Three evaluations of lumbar bone mineral content (BMC) were carried out at different times at intervals of a few days in 10 normal volunteers aged between 18 and 60. The dual photon absorptiometry technique was employed (using gadolinium-153 as radioactive isotope). Data on 20 subjects aged from 23 to 62 were recorded on magnetic tape and processed ten times by the same operator. The data for individual subjects proved satisfactorily reproducible and the repeated processing of a single scan by the same investigator gave very satisfactory results. The findings demonstrate that the evaluation of lumbar BMC by dual photon absorptiometry can provide highly reproducible and acceptably accurate data.

Adult↗
PubMed1992

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.

Base Sequence↗
PubMed1987

Use of monoclonal antibodies for diagnosis and treatment of liver tumours.

Antibody-guided diagnosis can provide information regarding malignant disease which is not obtainable by conventional techniques and in some instances this approach can help to achieve prolonged tumour-free survival. Nevertheless, there are many technology improvements that need to be made if this method is to be widely applied for routine diagnosis and therapy. Already, encouraging therapeutic responses have been reported using 131I-labelled polyclonal and monoclonal antibodies in the treatment of primary and metastatic liver tumours. It is essential, however, to improve the radiolabelling of monoclonal antibodies for therapeutic use. It would be more convenient and effective to use radioactive isotopes which are pure beta emitters, e.g. 90Y and 32P which would allow for outpatient therapy. It is essential to evaluate the use of multiple antibodies or antibodies that bind to multiple antigenic targets within tumours to improve dose rate and total dose amplification. Finally, encouraging clinical pilot studies should be followed up by documented randomized clinical trials in order to define properly the clinical place of monoclonal antibodies, in both the diagnosis and the therapy of malignant disease, including primary and metastatic liver tumours.

Antibodies, Monoclonal↗
PubMed1996

The aphid sex pheromone cyclopentanoids: synthesis in the elucidation of structure and biosynthetic pathways.

Identification of a range of aphid sex pheromones as comprising the cyclopentanoids (4aS,7S,7aR)-nepetalactone, (1R,4aS,7S,7aR)-nepetalactol and the (1S)- and (1R,4aR,7S,7aS)-nepetalactols required samples authenticated by 1H and 13C NMR. These and related compounds were provided by small scale synthesis and extraction from plants in the genus Nepeta (Lamiaceae). The subsequent discovery that the synthetic sex pheromones could attract males, and also parasitic wasps that attack aphids, has created a need for large scale syntheses of the cyclopentanoids. This is afforded by cyclisation of the 8-oxo-1-enamine of citronellal as originally developed by Schreiber and co-workers (1986). Investigation into the biosynthesis of the cyclopentanoids by plants for exploiting aphid sex pheromones in crop protection by means of molecular biology required synthesis of putative biosynthetic intermediates, some with radioactive isotopic labelling, particularly 8-oxidised monoterpene alcohols and aldehydes.

Animals↗
PubMed1994

Recent developments in medical applications of SIMS microscopy.

The purpose of this review is to present the recent developments in the medical applications of SIMS microscopy. This technique is one of the microanalytical mass spectrometry methods which allow in theory the detection of all the elements of the Mendeleiev table as well as the separation of stable and radioactive isotopes. It is based on a phenomenon whereby a biological sample surface is sputtered by bombardment with an energetic 'primary ion' beam. Part of the sputtered matter is ionized and the resulting 'secondary ions' are characteristic of the atomic composition of the analyzed area. These secondary positive or negative ions are collected and separated in a mass spectrometer at low or high mass resolution, which is dependent on both the element studied and its concentration. An analytic image which conserves the tissue distribution of the selected element is displayed on a fluorescent screen linked to an image processing system. Local elemental concentration can also be measured. Results are highly dependent on the techniques used for sample preparation which should preserve both the chemical and the structural integrity of the tissue. Further, the ionic images must be correlated with corresponding images of the same areas of the serial sections observed in a photonic microscope. With our SIMS microscope (lateral resolution approximately 0.5 microns, and mass resolution 300 to 12,000) we have demonstrated that this microscopic imaging technique is suitable for physiopathological studies. We revisited thyroid iodine metabolism by mapping chemical elements such as 32S and 127I, characteristic of hormonal physiology. Newly organified iodine (radioiodine) can be evaluated in relation to previously stored iodine (127I) in a given follicle, thus allowing an appraisal of glandular adaptation to aging and iodine overload. Another area in which SIMS can be used in medicine, is for the localization of drug markers in tumor tissue (e.g. fluorine-5-fluouracil, iodine in iododeoxyrubicin). This could facilitate the evaluation of the intratumor drug concentration at the onset of the treatment. Likewise, SIMS can be used to localize radiopharmaceuticals used in diagnosis (e.g. technetium) and therapy (131I of metaiodobenzylguanidine). This would permit a better evaluation of the radiation dose delivered to tissue. Further prospects are within reach with the imminent advent of higher lateral resolution (0.05 microns) SIMS microscopes.

3-Iodobenzylguanidine↗
PubMed2006

A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin.

Pseudomonas exotoxin A (PE) inhibits protein synthesis by NAD-dependent ADP-ribosylation of eukaryotic elongation factor 2. Traditionally, toxin activity has been characterized, either in living cells or cell-free systems, using radioactive compounds for quantification. The increased costs of radioactive waste disposal together with heightened security concerns have made the use of radioactive isotopes less attractive for routine laboratory assays. We therefore adapted a cell-free rabbit reticulocyte in vitro transcription-translation system that utilizes a reporter (beta-galactosidase) to measure toxin activity. The assay for PE is rapid, scalable, log-linear, NAD dependent and can be used to assess the neutralizing activity of anti-PE antibody preparations.

Adenosine Diphosphate Ribose↗
PubMed2004

Separation of radionuclides in a multitracer prepared by thermal neutron fission of 235U.

In response to the needs of research groups in Japan, a chemical separation procedure was developed for isolating specific radioactive isotopes from a multitracer prepared by the reaction of 235U fission induced by thermal neutrons. This efficient technique is based on solvent extraction and separates the multitracer isotopes into six groups. It is intended to be used in an automated rapid extraction system.

Journal Article↗
PubMed2006

Gamma-ray imaging with a Si/CsI(Tl) Compton detector.

We present results from Compton imaging of gamma-ray sources using an instrument constructed from thin silicon scattering detectors and CsI(Tl) absorbing detectors. We have successfully imaged single and double point sources for several common radioactive isotopes ((137)Cs, (60)Co, (22)Na, (54)Mn). The measured angular resolution is 11.6( composite function) FWHM at 662keV. In parallel with the hardware effort, a GEANT4-based simulation code was developed. Comparisons between real and simulated data are discussed.

Cesium↗
PubMed2006

Influence of inlet positions on the flow behavior inside a photoreactor using radiotracers and colored tracer investigations.

The influence of the inlet positions on the flow behavior inside a photoreactor has been studied using colored tracer and radiotracer investigations. The photoreactor for water disinfection is a tubular reactor inside which UV emitting lamps are placed. The key to its good performance is a combination of optimum irradiation dose and fluid residence time distributions (RTD). The inlet system is one of the better possibilities of controling the fluid RTD. Three configurations have been tested. In the first step, colored tracer experiments help us to raise the mean trends of the flow behavior for the different configurations. When the inlet is located at the central bottom of the reactor, the flow is closed to a perfect mixing whereas lateral inlets lead to a plug flow with axial dispersion behavior. RTD measurements using radiotracer was carried out in a wide range of flowrates. The axial dispersion coefficient increases linearly with the Reynolds number. It is lower when the lateral inlet is divided in three inlets uniformly distributed around the bottom of the reactor. This paper shows the usefulness of radioactive isotopes as tracers in the field of wastewater treatment.

Coloring Agents↗
PubMed2005

Selenocysteine in proteins-properties and biotechnological use.

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

Biotechnology↗
PubMed2004

A method to quantitatively detect H-ras point mutation based on electrochemiluminescence.

Conventional methods for point mutation detection are usually multi-stage, laborious, and need to use radioactive isotopes or other hazardous materials, and the assay results are often semi-quantitative. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electrochemiluminescence (ECL) assay was coupled with restriction endonuclease digestion directly from PCR products. Only the wild-type amplicon containing the endonuclease's recognition site can be cut off, and thus cannot be detected by ECL assay. Using the PCR-ECL method, 30 bladder cancer samples were analyzed for possible point mutation at codon 12 of H-ras oncogene. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than three orders of magnitude. The point mutation was found in 14 (46.7%) out of 30 bladder cancer samples. The experiment results demonstrate that the PCR-ECL method is a feasible quantitative approach for point mutation detection due to its safety, high sensitivity, and simplicity.

Calibration↗
PubMed2004

Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter.

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.

Animals↗