Detection Of Small Amounts Research Articles

一般電球における酸素量の検出

一般電球における酸素量の検出

The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

The potential of an immunogold-silver staining for the study of human suspended Langerhans cells at the transmission electron microscopic level was evaluated. Cells were labeled, by using a preembedding technique, with 5-nm colloidal gold particles followed by silver enhancement. The use of small colloidal gold particles permits a detection of small quantities of antigen; the metallic silver deposition around gold granules gives rise to a large electron-dense marker which can be easily detected even at low magnification. Ultrastructural details were well preserved, and the background was not significant. The major advantage of the present immunogold-silver staining is that it enables to detect labeled cells easily, even when limited amounts of antigenic moieties are present on a low percentage of cells. Therefore, a rapid and simultaneous evaluation of both immunophenotype and ultrastructural details of investigated cells is allowed.

Spotgun-Applied Hexazinone: Release of Red Pine (Pinus resinosa) from Quaking Aspen (Populus tremuloides) Competition and Residue Persistence in Soil

Weed control and red pine release by spotgun-applied hexazinone in a northern Ontario plantation were evaluated 3 yr after treatment, while hexazinone residues and lateral movement in the sandy loam soil were determined 1 yr after treatment. Hexazinone, grid pattern spot applied at 480 mg ai/spot, approximating 1.6 kg ai/ha, resulted in 88% quaking aspen stem dieback and variable suppression of white birch and pin cherry. The height and basal diam of treated red pine were 131 and 150% of control, respectively, after 3 yr. Hexazinone residues were reduced to 1% at the treated spot and did not move laterally beyond 0.5 m, 1 yr after treatment. Detection of small amounts of metabolites A and B (0.2 and 0.3%) indicated the non-cumulative degradation of hexazinone in soils.

A new radioimmunoassay for the detection of small amounts of white cells and platelets in red cell concentrates: implications for blood transfusion

In the procedure for quality control of red cell concentrates, made white cell (WBC)-poor by filtration, the particle-counting technique was found to be insufficiently sensitive in detecting the remaining WBCs and platelets. Therefore, direct radioimmunoassays were developed using murine monoclonal antibodies specific for platelets, granulocytes, and T lymphocytes. The sensitivity for platelets was 40 x 10(3) per mL, that for granulocytes was 10 x 10(3) per mL (starting from 0.2 mL of red cell filtrate), and that for T lymphocytes was 0.006 x 10(6) per mL (starting from 5 mL) and 0.0015 x 10(6) per mL (starting from 50 mL). These direct assays were used in experiments on filtration with three types of filters: the Cellselect B-1005, B-1014 and the B-1013 (bedside filter). After filtration of 1 unit of blood cell suspension through the B-1005, the number of remaining platelets was found to vary between less than or equal to 0.04 x 10(6) and greater than 15 x 10(6) per mL (n = 16); after filtration through the B-1013 filter, the remaining platelets were greater than 0.04 x 10(6) per mL. Upon filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining platelets varied between less than or equal to 0.04 x 10(6) and 5 x 10(6). In both filter types, the number of remaining granulocytes was always less than 0.01 x 10(6) per mL. A study of T-lymphocyte contamination revealed that, upon filtration of 1 unit of blood through the B-1005, T-lymphocyte numbers were less than or equal to 0.0015 x 10(6) to 0.15 x 10(6) per mL (starting from 5 and 50 mL); upon filtration through the B-1013 filter, the number of remaining T lymphocytes varied between less than or equal to 0.006 x 10(6) and 0.2 x 10(6) per mL (starting from 5 mL). After filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining T lymphocytes ranged from less than or equal to 0.006 x 10(6) to 0.1 to 0.5 x 10(6) per mL (starting from 5 mL). The direct radioimmunoassay is an improvement over the present electronic particle-counting techniques with regard to both sensitivity and specificity and may therefore be useful in quality control procedures in blood transfusion as well as in the development of new filters.

The immunogold-silver staining approach in the study of lymphocyte subpopulations in transmission electron microscopy

The potential of immunogold-silver staining has been evaluated in immunoelectron microscopic studies of human normal peripheral blood lymphocyte subpopulations. The cells were labeled, before being embedded in resin, using 5 nm colloidal gold particles and this was followed by silver enhancement. The use of colloidal gold particles permits detection of small amounts of antigen; the silver intensification forms a sphere of heavy metal around the gold granule giving rise to an ultrastructural marker which can be easily seen even at low magnification. The ultrastructural details of the cells were well preserved and there was no significant background staining. The major advantage of the present IGS technique is that it permits a rapid and simultaneous evaluation of both the immunophenotype and the ultrastructural characteristics of cells.

Influence of molecular weight on the crystallization of poly(vinylidene fluoride)

Five samples of Poly (vinylidene fluoride) with a molecular weight from 45,000 to 260,000 were analyzed by means of FT-IR spectroscopy in order to study the influence of molecular weight $$\left( {\bar Mv} \right)$$ on the crystallization process. The spectra of films obtained from methylethylketone solutions revealed the prevalence of form II (α) in the case of lower $$\bar Mv$$ and of form III (γ) in the higher molecular weight samples. The amount of form III was found to increase as the Mv decreased and as annealing temperatures increased. The deconvolution technique of vibrational spectra allowed detection of small amounts of other forms accompanying the prevailing one.

Orofacial infection of athymic mice with defined mixtures of acyclovir-susceptible and acyclovir-resistant herpes simplex virus type 1

Infection of athymic mice with defined populations of acyclovir-susceptible (thymidine kinase [TK]-positive) and acyclovir-resistant (TK-deficient or TK-altered) herpes simplex virus type 1 strains was used to simulate herpetic skin disease of the immunocompromised host. In vitro characterization of the defined virus mixtures revealed that the dye uptake method was quite sensitive in the detection of small amounts (3 to 9%) of acylovir-resistant virus. Mice infected with homogeneous virus populations exhibited a good correlation between clinical response and the in vitro drug susceptibility of the infecting virus. Animals infected with defined mixtures of viruses exhibited varied patterns of infection and responses to acyclovir treatment. However, disease severity was useful in predicting the TK phenotype of virus recovered from lesions. Pathogenic, TK-altered virus was responsible for progressive disease in animals receiving low-dose (0.25-mg/ml) prophylactic acyclovir or high-dose (1.25-mg/ml) delayed therapy. Although this mutant was recovered infrequently, it was responsible for clinically significant disease in the animals from which it was isolated.

Assessment of vitamin D sulphate in human milk using desorption chemical ionization mass spectrometry.

Vitamin D3 sulphate (SD3) identification in human milk was obtained using Desorption Chemical Ionization (DCI). The chemical ionization reagent gas used was nitrogen, molecules were ionized when the emitter was heated. SD3 was obtained from lactarium human milk and purified by high-performance liquid chromatography (HPLC). A selected ion monitoring (SIM) measurement was carried out with typical ions, m/z 366 for SD3 and m/z 384 for parent vitamin D3, the intensity ratio (I366/I384) greater than 1 being related to the presence of the sulphoconjugated form of vitamin D3 in the sample analysed. The detection of small quantities of SD3 in human milk is possible using this technique.

Modification of a commercial preparative HPLC system allowing for UV detection of small amounts of materials with high extinction coefficients

A Waters Prep LC/System 500A was modified in order to allow separation of very small quantities of light-sensitive substances exhibiting very strong extinction coefficients (e∼105) from large quantities of other materials.

Ready detection of small amounts of lactulose in dairy products by thin-layer chromatography

Ready detection of lactulose in milk and in mixtures with other sugars is achieved by diluting the sample ten times with acetone and successive thin-layer chromatographic separation on silica gel-boric acid, developed with acetonitrile: water 50∶20. The method allows detection of up to 0.02% lactulose on total carbohydrates and permits a semi-quantitative estimation of lactulose in milk.

Angiotensin II stimulation of renal prostaglandin synthesis elevates circulating prostacyclin in the dog.

The ability of the kidney to release prostacyclin (PGI2) under direct stimulation with exogenous angiotensin II was studied in the pentobarbital-anesthetized dog. We assayed for prostacyclin-like activity in systemic arterial blood by continuously monitoring platelet aggregation in vivo and with the blood-bathed bovine coronary artery, which relaxes to prostacyclin. This parallel bioassay allows detection of small amounts of prostacyclin-like activity released into the systemic circulation. Angiotensin II, infused at a rate of 10 or 20 ng/kg/min into the renal artery inhibited latelet aggregation in 8 out of 18 dogs, relaxed the bovine coronary artery in 13 of 16 dogs, and lowered systemic arterial blood pressure 5-10 mm Hg in all but 3 animals. These effects of antiotensin II could be blocked by treating the animal with indomethacin (2 mg/kg, i.v.) and mimicked by administration of exogenous prostacyclin. Infusion of the same dose of angiotensin II (10 or 20 ng/kg/min) intravenously did not release prostacyclin; blood pressure increased during intravenous infusion, and platelet aggregation remained unchaged. These data are consistent with the hypothesis that prostacyclin, of renal origin in these experiments, can act as a circulating hormone. Prostacyclin should not be regarded only as an antiaggregatory substance, but also as a potentially important modulator of blood pressure.

Self-sandwich method. An improved immunoperoxidase technic for the detection of small amounts of antigens.

The unlabeled antibody peroxidase-antiperoxidase (PAP) sequence has been widely accepted as the most sensitive method for demonstrating antigens in paraffin sections. However, the results have been unpredictable in routine surgical specimens, with frequent false-negative stains. In order to amplify the staining reactions a self-sandwich method was used. The amplifier (antigen itself) was added after the application of specific antibody. The procedure may be repeated several times before the application of the bridge antiserum followed by PAP complexes. The method was designed to increase the number of antigen-antibody layers without increasing the number of heteroantisera in the system. Specimens derived from routine tonsillectomy were studied semi-quantitatively. Based on the number of positively stained Ig-containing cells in germinal centers, the sensitivity of the self-sandwich method was estimated to be 20 to 50 times that of the PAP method. In addition, extracellular Ig (reticular staining) and surface Ig were also stained positively in the germinal centers and lymphocytic mantles, respectively, but were not demonstrated by the PAP method. The enhancement of the sensitivity was achieved without compromising the specificity.

A microplate immunoenzyme assay for anti-influenza antibodies

An enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase is described for the detection and quantitation of anti-influenza virus antibodies. Compared with complement fixation and hemagglutination inhibition tests, ELISA is far superior with respect to sensitivity and reliability. Non-specific viral inhibitors present in sera do not affect the titer in ELISA. Its sensitivity, close to that of radioimmunoassay permits detection of small amounts of antibodies in pulmonary secretions and supernatants from in vitro spleen cell cultures.

Thrombin Generation and Neutralization Test (TGNT) - a Simple, Practical, and Sensitive Assay for Plasma Heparin Quantitation

A simple, quantitative method for detection of small amounts of heparin in human plasma is described. This method is based on the activation of coagulation factor X to its enzymatic form (Xa), by a mixture of RVV platelet substitute (Esnouf and Williams 1962, Esnouf and Jobin 1967) in a plasma rendered fibrinogen free without affecting other clotting factores (Bell et al. 1968, Bell 1973), by Arvin (Ancrod). Activation of factor X, results in thrombin generation. Thrombin inactivation by its natural inhibitors depends on the time elapsed from the start-point of activation. The inactivation process is enhanced by heparin (Biggs et al. 1970, Blombäck et al. 1963) and this enhancement depends on the quantity of heparin in the plasma.

The fluorescence-plaque test — A new sensitive technique for the detection of antigens on antibody plaque-forming cells

A new technique is described which allows the detection of small amounts of antigen on the membrane of antibody plaque-forming cells. The test system combines the indirect immunofluorescence test and the Jerne plaque technique, and is about 10 times more sensitive than the ordinary cytotoxic plaque technique.

Assessment of several pasture species, particularly tropical legumes, for oestrogenic activity

Several pasture species, mainly legumes of established or potential application to tropical and subtropical pastoral areas, have been assessed for potential oestrogenic activity. The major criterion of potency adopted was the change in the ratio of the concentration of nucleic acids (RNA/DNA) in the uterine tissue of ovariectomized ewes fed exclusively on the test species for 5 days; in one experiment, samples of uterine tissue were obtained by biopsy before and after the experimental treatment. Increases in uterine weight following five daily injections of 10 µg stilboestrol dipropionate or the ad libitum consumption of fresh red clover (Trifolium pratense) appeared to be comparable at c. 50%; both treatments increased the nucleic acid ratio by more than 100%. It was concluded that change in the nucleic acid ratio offered a much more sensitive technique for the detection of small amounts of oestrogen than the traditional uterine weight method; the response obtamed following the ingestion of red clover, a species known to be oestrogenic, demonstrated the applicability of the nucleic acid ratio technique for the detection of phyto-oestrogenicity. No evidence of oestrogenic activity was obtained from the tropical pasture species Desmodium intorturn, Lotononis bainesii, Macroptilium atropurpureum, Lablab puupureus, Trifolium semipilosum or Digitaria decumbens. The results obtained following the feeding of an experimental strain of creeping-rooted lucerne (Medicago sativa) merely suggested a slight potential for oestrogenicity.

A micro tissue culture test for the titration and neutralization of rubella virus

A simple and reproducible micro tissue culture assay has been devised in RK13 and LLC-RK1 rabbit kidney cells for the titration and neutralization of rubella virus. In this "one-step" assay all virus and serum dilutions were prepared with spiral loops in disposable microplates and tissue cultures suspended in medium 199 and 3% horse or fetal calf serum were added to the microcups simultaneously. Micro tissue cultures were kept in a humidified incubator (36 °C) under a constant pressure of 5% CO2 for 8 days and were read microscopically for viral cytopathic changes on the seventh and eighth day. The microneutralization test performed in LLC-RK1 cell cultures was shown to be a reliable method for the detection of small amounts of rubella antibodies in human sera.The micro assay may be also applied to the virus titration of live, attenuated rubella vaccines.

Enteric excretion of metabolites of steroid hormones in the human subject. IV. Isolation of 5-beta-pregnane-3-alpha, 20-alpha-diol from meconium.

Pregnanediol (3α,20α-dihydroxy-5β-pregnane) has been isolated and identified from pools of meconium excreted by male and female infants during the first 24 hr after birth. From 85 to 95% of the steroid was obtained from the sulfate fraction and 5–15% from the glucosiduronate fraction. The isolation of pregnanediol from meconium offers further evidence of fetal metabolism of this steroid, and of the importance of the role of the sulfate conjugate in fetal physiology. The detection of small amounts of pregnanediol in the glucosiduronate fraction indicates that pregnanediol may be a substrate of glucuronyl transferase in the human fetus.

Permanent sensitive stain for choline-containing phospholipids on thin-layer chromatograms

The phosphomolybdic blue stain for choline of Levine and Chargaff can be used for detection of small amounts of choline-containing phospholipids on thin-layer chromatograms if they are first fixed with polyvinyl propionate to permit a washing step.

A sensitive method for the detection of iodinated compounds in human plasma, and its applications in the study of thyrotoxicosis.

ABSTRACT Iodinated tyrosines were identified in human plasma in certain familial disorders, after the large doses of radiation given in some cases of carcinoma of the thyroid and, as previously reported by this department, in some, but not all cases of thyrotoxicosis. The evidence for this statement is further strengthened by the results obtained with a modification of a chemical method for the detection of small amounts of iodinated compounds which were separated by paper chromatography. This method depends essentially on the treatment of paper chromatograms with eerie sulphate-arsenious acid reagent and is a development of that described by Bowden et al. (Biochem. J. 59: 93, 1955). Using this technique alone, and in conjunction with scintillation counting, it was possible to study the plasma of patients before and after the administration of radioiodine. In this way a radiation effect on the thyroid may be excluded as the cause for the appearance of abnormal iodinated compounds in the plasma of certain ...