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At least 919 records · Page 51Linked to original sources
PubMed1988

"Run-down" of gamma-aminobutyric acidA receptor function during whole-cell recording: a possible role for phosphorylation.

When using whole-cell recording methods and a minimal intracellular medium containing only inorganic ions, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, we have observed a time-dependent decrease in the responsiveness of cultured chick spinal cord neurons to gamma-aminobutyric acid (GABA). The current evoked by 30 microM GABA progressively declined to approximately 30% of its initial value after five applications at 10-min intervals. This was accompanied by an equivalent decline in the GABA-evoked membrane conductance. "Run-down" of the response was reduced when Mg2+-ATP was present in the pipet solution. Inclusion of ATP-gamma-S, an analog that donates a thiophosphate group resistant to hydrolysis, also reduced run-down. The nonhydrolyzable analog beta, gamma-imidoadenosine-5'-triphosphate was without effect. These results suggest that an ATP-dependent process, possibly phosphorylation, is involved in the maintenance of GABAA receptor function.

Adenosine Triphosphate↗
PubMed1989

Turbidimetric latex immunoassay of placental lactogen on microtiter plates.

In this latex immunoassay for human placental lactogen, microtiter plates are used as the reaction vessel and the absorbance at 405 nm is measured to quantify the reactions. This 30-min assay necessitates only one serum dilution and two pipetting steps. The calibration curve extends from 0.5 to 15 mg/L. CVs range from 4.2% to 7.0% for within-run determination and from 7.0% to 11.2% for between-run determinations. A correlation coefficient of 0.949 was obtained for 84 sera when the method was compared with a commercial radioimmunoassay.

Female↗
PubMed1988

A simple assay based on HIV infection preventing the reclustering of MT-4 cells.

This report confirms and extends the recent work of Pauwels et al. on a "reclustering" assay (a simple microtitration plate method) for the determination of human immuno-deficiency virus (HIV) infection of MT-4 cells. MT-4 cells, which are highly susceptible to and permissive for HIV, typically grow in clusters. In the absence of virus these cell aggregates, after dissociation by pipetting, reform into clusters within 2 to 3 hours. Growth of HIV results in an inhibition of reclustering, with an end-point some 4-5 days after initiation of infection. In cultures inoculated with 5 to 8 TCID(50) of HIV, only 2-4% of the cells remain viable after 4 days. Correspondingly, HIV antigens can be detected by immunofluorescence in more than 90% of the cells remaining in the culture. The sensitivity of the "reclustering" assay is only slightly less than that of the immunofluorescence test. A colorimetric assay is also described that employs a tetrazolium salt (designated as MTT) to measure the cytolytic effect of various dilutions of HIV; comparable virus titres were obtained. This reclustering assay now appears to offer the simplest method for titration of prototype HIV in virus stocks and when used in drug evaluation tests and for measurement of HIV neutralizing antibodies.

Cell Aggregation↗
PubMedDate not supplied

Location dependent biases in automatic 96-well microplate readers.

Procedures performed in 96-well microplates and quantitated by automatic readers assume instruments to be precise, accurate, and free of well location dependent bias. Instrument specifications generally focus on precision and accuracy without specifically addressing biases which are dependent on well location. These biases appear to be meniscus dependent and can be demonstrated in varying degrees in automatic readers of many designs by using a reverse plate wet test, which compares repeated readings of a dye loaded plate in normal and reversed positions. This test analyzes differences between readings and is, therefore, independent of pipetting error or other experimental variables such as protein binding or immunoassay variability. Different plates increased or decreased the magnitude of observed errors but did not themselves cause the errors measured by the reverse plate wet test. Error patterns were consistent for each reader and varied widely among the 16 instruments tested. Only 4 of 16 instruments passed an existing manufacturer's specification for precision, and only one of the 16 readers tested passed a similar specification for accuracy. The severest location dependent bias was found in an instrument which exhibited excellent repeatability and consistently passed its built-in diagnostic tests. One reader with significant bias was returned to the manufacturer for routine service and calibration, but it was not demonstrably improved. The reverse plate wet test is an extremely useful diagnostic tool for quality control at all stages of instrument manufacture and use.

Evaluation Studies as Topic↗
PubMed1988

X-ray analysis of biological fluids: an update.

X-ray analysis has been used for some twenty years to quantify elements including Na, K, Cl, Ca, Mg and phosphorous in biological fluid samples. The method consists of accurate pipetting of 50-150 pl samples and appropriate drying to form crystalline deposits. Quantitation is obtained by comparison of x-ray intensities with comparable standard crystalline deposits. Electron microprobe analyses have contributed much to our understanding of renal transport function including physiological controls and pathological influences on the tubular handling of the major elements. New approaches to x-ray analysis of solid tissue will further our knowledge concerning transport properties in the kidney and other tissues.

Animals↗
PubMed1988

Evaluation of four commercially available assays for free thyroxin.

We assessed two two-step and two analog assays for measuring free thyroxin (FT4) in serum: Clinical Assays' "GammaCoat Free/Total T4" (CA), Vitek's "KinetiCount Phase II Free T4" (VTK), Diagnostic Products Corporation's "Coat-a-Count Free T4" (DPC) kit (June 1987 version), and Amersham's "Amerlex-M Free T4" (AMX). The VTK assay is automated except for the initial pipetting step. Interassay results correlated well except for samples with abnormal serum albumin concentrations. FT4 values for hypoalbuminemic samples showed a highly significant (P less than 0.0001) correlation with serum albumin concentration in the DPC and AMX assays. The relationships are described by the equations y = 0.382albumin (g/L) + 0.81 pmol/L and y = 0.450albumin (g/L) - 3.20 pmol/L, respectively. When we used an equation derived from the Law of Mass Action to adjust FT4 values to values expected at an ideal albumin concentration, the observed correlation of albumin and FT4 was abolished completely in the DPC assay, and partly so in the AMX assay. The precision of CA was comparable with that of the analog assays; the CV for the VTK assay was approximately twice that for the other three assays.

Humans↗
PubMed1977

Assay of phenobarbital: adaption of "EMIT" to the centrifugal analyzer.

We have adapted the centrifugal analyzer to the homogeneous enzyme immunoassay ("EMIT") for phenobarbital. The assay was modified to give greater range and sensitivity, and less reagent is needed. The transfer disc is prepared with totally automatic pipetting. Results are calculated with a micro-computer that provides a logit transformation of absorbance data. Precision and accuracy are excellent, and results correlate well with those by gas-liquid chromatography.

Autoanalysis↗
PubMed1986

Automated office hematology instruments.

Evaluation of an automated hematology instrument begins with analysis of one's practice needs, followed by analysis of initial start-up costs, cost of maintenance, and staffing. In one situation, the decision to purchase an automated instrument may allow personnel working with low-efficiency pipetting and slide chamber counting time to assist in other functions of the clinic. In another practice, an automated counter may permit in-office complete blood counts to be added to the workload of current staff. These instruments are more than time-saving conveniences. They can facilitate clinical decision making and monitoring for complications of disease and therapy by making it easier for the physician to obtain reliable hematologic results. They can generate additional practice income and reduce patient cost by improving the quality and efficiency of medical care.

Automation↗
PubMed1978

An improved technique for immunofixation of electrophoretograms.

Immunofixation coupled with electrophoresis is winning favor as an easy, sensitive, and versatile method for detecting specific protein fractions. Here, we discuss problems which appear when antisera are applied onto the electrophoretic medium (Cellogel or cellulose acetate). Longitudinal slits can be made in the electrophoretic medium without worsening resolution. Such longitudinally slitted electrophoretic membranes can be used in electrophoresis and specific antisera applied simply by pipetting them onto the electrophoretograms. The method has been used successfully in typing immunoglobulins.

Blood Proteins↗
PubMed1986

Multicentre evaluation of an enzymatic method for creatinine determination using a sensitive colour reagent.

A new enzymatic colorimetric method for the determination of creatinine in serum and urine (Creatinine PAP, Cat. No. 839434 and 836885, Boehringer Mannheim GmbH, Mannheim, FRG) was evaluated in 16 clinical chemistry laboratories and the manufacturer's testing laboratory. The test is based on the enzymatic degradation of creatinine and its reaction products by creatininase, creatinase and sarcosine oxidase. The H2O2 produced by the oxidation of sarcosine is determined using a modified Trinder reaction. The test can be carried out either manually or in mechanized analysers which enable the pipetting of a starter reagent to be made. The following results were obtained: Depending on the analyte concentration (range 40 to 1240 mumol/l), medians for the coefficients of variation were: 4.6-0.9% within-run and 6.4-2.8% between-day. At 546 nm the linear measuring range extended from 13 mumol/l (detection limit) to 1780 mumol/l, at 510 nm from 9 to 890 mumol/l. Recoveries in aqueous and human serum based standards as well as method comparisons with Fuller's earth methods and an enzymatic UV test indicate a high accuracy of this new enzymatic method in serum and urine. No interference was observed with haemolysed and lipaemic sera. This also applied to anticoagulants and to 36 drugs at therapeutic concentrations, with the exception of calcium dobesilate, which led to decreased values. Icteric samples containing 120-310 mumol/l bilirubin invariably led to decreased creatinine values (10-50 mumol/l lower). In a collaborative study substantially better interlaboratory agreement was observed with the new colorimetric enzymatic test than with the comparison methods (enzymatic UV test and various Jaffe procedures). In conclusion, this new enzymatic colorimetric test permits a precise and specific determination of creatinine in serum and urine. It makes a considerable contribution to improving the interlaboratory comparability of creatinine determinations and is suitable for routine use.

Bilirubin↗
PubMed1985

[Micromanipulatory sperm injection--a new method in the treatment of infertile males].

Three couples were selected for this preliminary sperm injection study with their consent, since in all three cases the zona free hamster egg penetration test was negative and in one couple previous attempts of in vitro fertilization were unsuccessful. Of 22 eggs, recovered from three patients, 9 from each patient 3 were injected with sperm, while the others were inseminated by the standard IVF-protocol. Before manipulating human eggs extended experiments were performed with animals eggs, so decreasing the rate of damage to under 10%. Additionally we optimized the method by using an individual adapted equipment. Manipulation was performed by penetration of the zona pellucida with a 5 to 7 mu thick injection pipet into the perivitilline space so damaging of the egg cytoplasm was avoided. This special technique for sperm injection was not used till now in this indication. From the nine eggs used in this experiment one egg developed a pronucleus and one egg developed to a four-cell stage. We could not achieve a pregnancy.

Embryo Transfer↗
PubMed1985

Quantification of human serum apolipoprotein AI by enzyme immunoassay.

We developed a quantitative assay for apolipoprotein AI (apo AI) in human serum, using a "sandwich"-type enzyme-linked immunosorbent assay. Diluted serum samples were pipetted into the wells of polystyrene microtiter plates that had been previously coated with purified rabbit anti-human apo AI antibodies. After incubation for 2 h and washing, antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) were added and incubated for 2 h; after further washing, the bound enzyme was assayed by oxidation of o-phenylenediamine. Assay conditions were optimized for the incubation time and the amounts of coating antibodies and conjugate. Assay sensitivity is about 0.5 ng of apo AI, with a working range of 1 to 14 ng, similar to that of radioimmunoassays for human apo AI. The standard curves for apo AI in serum or HDL and for purified apo AI were parallel. Delipidation, heat treatment, or addition of detergents did not affect the amount of immunoassayable apo AI in human serum. The intra- and interassay CVs were 4 and 8%, respectively. Results for 100 serum samples compared well with those by immunonephelometry (r = 0.94).

Apolipoprotein A-I↗
PubMed1979

Microcomputer controlled titration for determination of protein nitrogen in feeds and wheat.

The determination of protein nitrogen in feeds and wheat by microcomputer controlled titration is described. The method involves direct titration of ammonia with standard hypochlorite titrant in the presence of bromide. The titrant is delivered by an automatic buret, and the microcomputer controlled, automatically computed potentiometric end points are precise to 0.1% over a 5-fold concentration range of nitrogen. Digestions performed with both mercury and copper catalysts show comparable results. Samples are weighed before digestion by an electronic balance interfaced to the computer which records sample number and weight. An automatic pipet aliquots, dilutes, and buffers samples directly from the digestion tubes; the samples can be immediately titrated with the automatic titrator. The results for protein in NBS standards and check feed samples from an offical testing program compare closely with average values reported for these standards. Results show that feed and wheat samples contained 10-100% protein. Precision for successive aliquots of the same digests is 0.1-0.4%relative standard deviation; precision for multiple digestions of the same sample is 0.1-0.8%.

Animal Feed↗
PubMed1985

[Rapid method for evaluating intra-erythrocyte Na+ activity].

The measurements of intracellular "Na+ activity" was performed in 10 ml of heparinized venous blood. First the blood was three times washed in isotonic magnesium chloride solution (114 mmol/l). Thereby the buffy coat was removed. Then the microhematrocrit was taken for packet cell volume determination. After the erythrocytes were lysed by ultrasound. Sodium "Na+ Activity" is measured in the hemolysate by Ion-Selective electrode. With this method all "pipetting" operations are eliminated and for the "Na+ activity" determination was used ion-selective electrode with an indirect measurements, which is less influenced by the matrix. Reference intervals determined for a healthy population were 7.3 +/- 0.6 mmol/l.

Adolescent↗
PubMed1985

Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.

We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.

Allergens↗
PubMed1977

Triiodothyronine uptake assay with immobilized triiodothyronine antibody as the bound-free separating agent.

Available systems for evaluation of degree of unsaturation of thyroxine-binding globulin are hampered at the bound-free separation step. In current assays, inorganic sorbants or ion-exchange resins are used to separate free 125I-labeled triiodothyronine from that bound to thyroxine-binding globulin. The techniques are laborious, time consuming, and not readily adaptable to total automation. In the assay we describe, triiodothyronine antibody, immobilized on the walls of polypropylene test tubes, is used as the bound-free separating agent in the evaluation of degree of unsaturation of thyroxine-binding globulin. The assay is simplified to four steps; washing, centrifugation, and use of columns are eliminated; and the procedure is readily automatable with existing pipetting equipment. Correlation with existing methods is excellent.

Animals↗
PubMedDate not supplied

Enzymatic-ultraviolet determination of L-citric acid in wine: collaborative study.

A collaborative study was carried out on an enzymatic method for the determination of L-citric acid in wine, using the enzymes citrate lyase, malate dehydrogenase, and lactate dehydrogenase and the coenzyme nicotinamide-adenine dinucleotide phosphate. The study was performed by 18 laboratories using 4 blind duplicates of commercial wine. The method is simple and shows good precision. Coefficients of variation (CV) for reproducibility ranged from 1.8 to 3.4%; CVs for repeatability ranged from 0.76 to 2.62%. Analysts are cautioned to check the linear absorbance response of their spectrophotometers when performing this assay and also to take care in pipetting the relatively small volumes used in this procedure. The method has been adopted official first action.

Citrates↗
PubMedDate not supplied

Disposable cartridge extraction of retinol and alpha-tocopherol from feedstuffs.

Automated determination of fat-soluble vitamins by modern methods such as liquid chromatography is hampered by the initial extraction step. A simple technique is proposed that allows an appreciable increase in the actual rates of determination. Feedstuff samples are first hydrolyzed in an aqueous alcohol (mainly methanol)-potassium hydroxide solution. Instead of extracting retinol and alpha-tocopherol from the hydrolysis solution by an organic solvent, an aliquot of the solution is mixed with a small volume of a strong antioxidant solution (ascorbic acid) and pipetted onto a kieselguhr disposable cartridge where it is adsorbed. Retinol and alpha-tocopherol are eluted with isooctane at normal pressure. The proposed method has been compared with conventional techniques on many feed samples.

Animal Feed↗