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PubMedDate not supplied

[Prevalence of Mycoplasma orale as a contaminant of cell cultures in Argentina].

Over a period of 4 years 200 cell cultures were analysed for the presence of mycoplasma. Cultures were established cell lines from different origins, namely monkey, mouse and human, hybrid cell cultures and primary cultures. The cultures belonged to various research and industrial laboratories located in different areas of the country. Seventy per cent of investigated cultures were found to be contaminated with mycoplasma using a DNA fluorescent stain. Fifty cultures, selected at random out of the contaminated cultures, were further investigated to identify the prevalent serotype. For that purpose immunofluorescent reactions were performed using immune sera raised against several mycoplasma strains routinely found among contaminated cultures. Forty one cultures were contaminated with a single type of mycoplasma, whereas in the remaining nine, two or even three serotypes were detected. Mycoplasma orale II contaminated 40% of single infected cultures, followed by M. hyorhinis and A. laidlawii-A (12% each), M. arginini (5%), M. orale III (8%), A. laidlawii-B (2%). We were unable to serotype the remaining positive cultures, because of the lack of a full battery of immune sera against all known serotypes. The prevalence of M. orale in mycoplasma contaminated cultures thus far tested, indicates that human handling would be the main source of infection. This situation could be modified by avoiding mouth pipetting and adopting good microbiological techniques.

Acholeplasma↗
PubMed1991

Gingival crevicular fluid plasmin activity in different clinical conditions and after periodontal treatment.

Plasmin is an important enzyme in the process of wound healing and it has been suggested that it plays a role in the pathogenesis and healing of periodontal diseases. In this study, plasmin activity in crevicular fluid (CF) was measured in different clinical conditions and after periodontal treatment. 152 CF samples were collected with paper strips from 12 subjects. Five other subjects received a single periodontal treatment after the first sample collection and the post-treatment samples were collected 2, 5, 10, 20 and 40 days thereafter. The individual strips were placed immediately on plasminogen-free fibrin plates or in plastic vials containing sterile saline. The vials were agitated and 20 microliters of each solution was pipetted onto fibrin plates. The remaining solution was used in protein analysis. The plates were incubated for 24 hrs at 37 degrees C and the activity of plasmin determined by weighing the fibrin liquified by the samples. The variation of CF plasmin activity in different pockets within individuals was larger than between the subjects. There was a weak positive correlation between CF plasmin activity and the amount of plaque, bleeding tendency, pocket depth and bone loss by x-ray. However, plasmin concentration (units of plasmin activity per CF volume) did not correlate with clinical parameters. A weak negative correlation between plasmin activity per volume and the amount of CF was seen. Neither did CF protein concentration correlate with the clinical parameters. Periodontal treatment resulted in a dramatic decrease in plasmin activity. In 20 days the activity returned to pre-operative levels in four subjects out of five.

Adult↗
PubMed1991

Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

Binding, Competitive↗
PubMed1991

Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer.

We describe a new enzymatic assay for phospholipids that is rapid, sensitive, convenient, and inexpensive. The method is based on the fluorometric detection of H2O2, generated by the choline oxidase-catalyzed oxidation of choline, after liberation of choline from phospholipids by phospholipase D. Significant advantages over existing methods are that the entire reaction sequence can take place in a single vessel, a 12 x 8 well microtiter plate, and that the fluorescence intensity can be measured automatically with a Fluoroskan II (Labsystems Oy) detector. Extracting samples with organic solvents is unnecessary, although the method can be applied to extracts in isopropanol. The assay is approximately 60-fold more sensitive and has a limit of detection eightfold lower than currently available enzymatic colorimetric methods. Including solvent blanks, eight standards, and three quality-control pools, 34 samples can be pipetted and assayed in duplicate in 60 min. Results obtained by this procedure for total phospholipid choline in lipoproteins in primate plasma agreed well with those obtained for inorganic phosphorus by the Bartlett acid-digestion procedure.

Animals↗
PubMed1991

Rapid re-amplification of PCR products purified in low melting point agarose gels.

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.

Electrophoresis, Agar Gel↗
PubMed1991

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template.

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.

Bacteriophages↗
PubMed1977

A simplified determination of the urinary luteinizing hormone surge using the hemagglutination inhibition test.

The hemagglutination-inhibition test for urinary luteinizing hormone (LH) has been modified so that the test for LH surge can be assayed in 1 day. A first morning voided urine specimen submitted to the laboratory can be analyzed in 5 hours by ordinary bench procedures. This modification involves a 20-fold reduction, as compared with the older procedure, of the duration of incubation of the urine-antiserum mixture. Another modification is the choice of aliquots which are adaptable to automatic pipetting. Only a single dilution of urine is necessary to determine LH surge for the anticipation of ovulation. Daily LH determinations on 12 normally menstruating women (av. cycle 28.18 days) reveal consistent patterns. For the 40 cycles studied LH surge occurred 14.29 days (av.) prior to the onset of the next menstruation and coincided with the rise in basal body temperature. One woman with an unusually high incidence of familial twinning showed two large LH surges in each consecutive cycle.

Adult↗
PubMed1990

[In vitro separation and development of sheep blastomeres].

Four cell embryos collected by laparatomy from Sardinian breed ewes superovulated with FSH-p (16 mg Sigma), were divested of their zonae pellucidae (ZP) by micromanipulation or chemical methods (pronase 0.5%, tyrode pH 2.2). The blastomeres were separated by pipetting using a flame polished pasteur pipette in a Ca free medium (PBS. Sigma) and were inserted into previously evacuated Z.P. using a Leitz micromanipulator. The Z.P. were removed either mechanically or with acid tyrode; pronase was unable to digest them after incubation at 30 degrees C for 120 minutes. The single blastomeres were cocultured on a monolayer of ovine oviductal epithelial cells in TCM 199 + 10 FCS at 38 degrees C in 5% CO2 for 60 hours. No developments were observed in blastomeres obtained by acid digestion of the ZP while 50% of the other blastomeres continued their development until the 16 cell stages. Our results suggest that coculture with oviductal epithelial cell monolayers can support in vitro development of single ovine blastomeres.

Animals↗
PubMed1990

Automated blood-sample handling in the clinical laboratory.

The only significant advances in blood-taking in 25 years have been the disposable needle and evacuated blood-drawing tube. With the exception of a few isolated barcode experiments, most sample-tracking is performed through handwritten or computer-printed labels. Attempts to reduce the hazards of centrifugation have resulted in air-tight lids or chambers, the use of which is time-consuming and cumbersome. Most commonly used clinical analyzers require serum or plasma, distributed into specialized containers, unique to that analyzer. Aliquots for different tests are prepared by handpouring or pipetting. Moderate to large clinical laboratories perform so many different tests that even multi-analyzers performing multiple analyses on a single sample may account for only a portion of all tests ordered for a patient. Thus several aliquots of each specimen are usually required. We have developed a proprietary serial centrifuge and blood-collection tube suitable for incorporation into an automated or robotic sample-handling system. The system we propose is (a) safe--avoids or prevents biological danger to the many "handlers" of blood; (b) small--minimizes the amount of sample taken and space required to adapt to the needs of satellite and mobile testing, and direct interfacing with analyzers; (c) serial--permits each sample to be treated according to its own "merits," optimizes throughput, and facilitates flexible automation; and (d) smart--ensures quality results through monitoring and intelligent control of patient identification, sample characteristics, and separation process.

Blood Specimen Collection↗
PubMedDate not supplied

A comprehensive gas chromatography procedure for measurement of drugs in biological materials.

Procedures have been developed for the determination in serum and urine of 21 drugs which include all of the barbiturates, methyprylon, glutethimide, meprobamate, propoxyphene, methaqualone, primidone, diphenylhydantoin, methadone, codeine, morphine, amphetamine and methamphetamine. The method involves pipeting 1.0 ml of serum or urine into an extraction tube, adding an internal standard together with either a pH 4.9 or pH 8.3 buffer and extracting with chloroform. The drugs in the extract are concentrated by exaporation of the organic layer and identified and quantitated by gas-liquid chromatography (GLC) using a column packed with 3 percent OV 17. GLC analysis times are kept below seven minutes by using temperature programming which allows good separations of drugs of widely varying volatilities. All calculations are based on peak areas determined by an electronic integrator. Losses of drugs during extraction, evaporation and chromatography steps are compensated for by the use of internal standards. Actual extraction efficiencies of drugs from serum or urine range from 75 to 100 percent although recoveries, based on standards carried through the entire procedures, are quantitative. Precision (s.d.) based on replicate determinations is approximately plus or minus 0.2 mg per dl.

Alkaloids↗
PubMed1990

Evaluation of a rapid method for measuring theophylline in a clinical setting.

The need to determine rapidly the theophylline levels of patients in ambulatory settings such as family practice offices has led to the development of instruments purported to be fast, reliable, and cost-effective. This study evaluated one such instrument, the Ames Seralyzer, and compared the findings with those of the Du Pont aca. Forty-six patient samples were split and run on both instruments by trained laboratory personnel. Validation studies yielded a correlation coefficient of r = .9680 (N = 46). Precision assays showed the 27.5 and 82.5 mumol/L (5 and 15 mg/L) levels of theophylline found in control samples produced day-to-day coefficients of variation of 8.8% (n = 43) and 5.8% (n = 35), respectively. After initial evaluation of the Seralyzer, duplicate assays were performed because of erratic results. The evaluators felt that a major source of error was in the diluting and pipetting steps involved in the procedure. The Seralyzer was easy to run but did require some technique-dependent skills.

Ambulatory Care↗
PubMed1990

Automated quantification of creatine kinase MB isoenzyme in serum by radial partition immunoassay, with use of the Stratus analyzer.

We evaluated the analytical and clinical performances of a new radial partition immunoassay for measuring the mass concentration of creatine kinase (CK)-MB in serum. All pipetting, washes, incubations and data reduction were performed in 8 min by the Stratus (Dade) fluorometric analyzer. Within-assay and between-assay CVs were respectively 5.5% and 8.4% at 21 micrograms/L, and 4.2% and 3.4% at 48 micrograms/L. Assaying serial dilutions of serum samples with high CK-MB concentrations demonstrated excellent linearity. Results of the Stratus technique correlated well (n = 115, r = 0.98) with those of the Tandem-E CKMB II assay. There was no interference from hemolysis, bilirubin, rheumatoid factor, or added CK-MM (up to 3500 U/L); consequently, CK-MB can be determined in undiluted serum, even in the presence of high total CK activity. The mean CK-MB concentration in 105 blood donors was 1.9 (SD 1.3) micrograms/L. For seven myocardial infarction patients who received prompt fibrinolytic therapy, the mean CK-MB concentration was 4.5 (SD 1.8) micrograms/L at admission, and maximum concentrations, 119 (SD 94) micrograms/L, were recorded 16 h later. CK-MB returned to concentrations less than 10 micrograms/L within 72 h.

Acute Disease↗
PubMed1987

Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

Animals↗
PubMed1988

The 'tape-method': a new and simple method for quantitative culture of Pityrosporum yeasts.

A new method for quantitative culture of Pityrosporum yeasts, the 'tape-method', is presented. Samples for culture were taken from the skin by stripping with 1 cm2 of tape, whereafter the tapes were placed over a drop of sterile olive oil which was pipetted on a Sabouraud medium. Plates were incubated at 37 degrees C, and after 7 days the numbers of Pityrosporum colonies growing under the tapes were counted. With this method a difference in numbers of Pityrosporum colonies between seborrheic dermatitis and normal skin could be discerned. This difference was significant. It appeared that two successive strippings were sufficient for quantitative culture. The 'tape-method' appeared to be a reliable and inexpensive diagnostic tool.

Humans↗
PubMed1989

Effect of adhesive thickness on the shear bond strength of Scotchbond 2/Silux to dentin.

Seventy-five extracted human permanent molars were embedded in brass cups with cold-cure acrylic. Immediately prior to the preparation of a test specimen, the occlusal surface was ground wet on 180 followed by 600 grit SiC to expose dentin. The Scotchprep Dentin Primer was applied to the dried dentin surface with a brush for 60 seconds, and dried with compressed air for 15 seconds. The tooth was mounted in a special device containing a split Teflon mold with a circular hole, 3.5 mm in diameter and 5 mm deep, and A:0.5 microL (50 microns), B:1.0 microL (100 microns), C:2.5 microL (250 microns), D:5 microL (500 microns) or E:10 microL (1000 microns) Scotchbond 2 pipetted into the teflon mold. The adhesive was cured by visible light for 30 seconds. Three increments of Silux were transferred to the Teflon mold, compressed firmly, and each cured for 30 seconds. The test specimens were disassembled 15 minutes after cure and stored in distilled water at 37 degrees C for 24 hours. A shear load was applied via a knife-edge rod in an Instron machine. The shear bond strength was expressed in MPa. The data were analyzed by ANOVA and Duncan's multiple range test. The mean (+/- SD) of the shear bond strengths were: A: 8.8 (3.5), B: 7.9 (2.7), C: 6.8 (2.5), D: 8.1 (4.2), E: 5.3 (2.4). Adhesive thickness from 50 microns to 500 microns (A, B, C and D) had no significantly different effect on shear bond strength. An adhesive thickness of 1000 microns resulted in a significant reduction in shear bond strength.

Bisphenol A-Glycidyl Methacrylate↗
PubMed1989

Transfer of the adenosine deaminase (ADA) gene of a B-lymphoblastoid cell line (LCL) to an ADA-deficient LCL by a microcell-mediated chromosome transfer technique.

As a model of somatic cell gene therapy, a normal adenosine deaminase (ADA) gene was introduced into a B-lymphoblastoid cell line (LCL) established from a patient with ADA deficiency by microcell-mediated chromosome transfer (MMCT). A LCL derived from his mother was used as a gene donor. Seven fusion experiments were performed and hybrid cells were pipetted into 123 wells. After selection in the presence of deoxyadenosine, cells grew in 12 wells at Week 9 after fusion. Among these wells, ADA activity of hybrids was low in 4 wells, 130-280% of that of the donor LCL in 7 wells and very high in one well. Hybrid cells in 4 wells with ADA-positive cells were investigated for the time-course of expression of ADA activity. In one well, ADA activity was expressed until Week 36, while, in 3 wells, ADA activity decreased or was lost between 21-36 weeks after fusion. These findings indicate the transfer of chromosome 20 containing a normal ADA gene into recipient cells and the deletion of this chromosome from some part of the hybrid cells. Karyotyping at Week 35 or 37 revealed 47 chromosomes in about 30% of the cells in 2 wells, which suggests that these hybrids were relatively stable in culture.

Adenosine Deaminase↗
PubMed1989

Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation.

Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data.

Automation↗
PubMed1989

Multilayer fluorescent immunoassay technique.

We describe a new multilayer immunoassay element for the determination of haptens in undiluted serum and plasma. Polysaccharide layers are coated onto a plastic base. The signal layer contains an immobilized antibody and a fluorescent-labeled hapten. A second layer, containing a pigment, acts as an optical screen. Sample spreading is achieved by a molded grid in contact with the upper layer of the immunoassay element. After sample is added to the element, endogenous analyte competes with the labeled hapten for the binding sites of the immobilized antibody; equilibrium is reached in 4-12 min. Because of the relative liquid-holding capacities of the layers and the grid, only a small amount of the free components remains in the signal layer. The signal is measured by front-surface fluorimetry. This technology has been applied to theophylline and thyroxin assays. Within- and between-run CVs range from 3% to 6%. Comparisons with fluorescent polarization immunoassays (Abbott TDx) showed excellent correlation (theophylline: r = 0.98, slope = 1.07, intercept = 0.3; thyroxin: r = 0.97, slope = 0.91, intercept = 0.8). The new method requires only one pipetting step (sample delivery) and is potentially applicable to a wide range of analytes.

Fluorescence Polarization↗