Immobilized enzymes in biochemical analysis.
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Amino Acids↗
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Appropriate technology for the quantitative assessment of the final reaction product of histochemical techniques.
This paper describes instruments for microfluorometry and microspectrophotometry, an interactive graphic tablet linked to a microprocessor for semi-automated morphometry, and systems using rapid scanning stages or television scanning techniques for automated analysis of biological material. Some applications of these techniques in clinical work are described.
Animals↗
Automated rheumatoid factor assay in a population of patients with rheumatic disease.
Rheumatoid factor has been measured in 128 patients having rheumatic disease to compare the Singer-Plotz assay with an automated particle size assay. Correlation coefficient between the two methods using regression analysis revealed r = 0.89, P less than 0.00001 when two different commercial sources of sensitized latex particles were used. Thus rheumatoid factor may be quantitated electronically and represented in arbitrary units (MPRF) without altering clinical interpretation. This methodology may be applicable to many other immunologic assays.
Autoanalysis↗
A survey of more than 90 commercially available luminometers and imaging devices for low-light measurements of chemiluminescence and bioluminescence, including instruments for manual, automatic and specialized operation, for HPLC, LC, GLC and microtitre plates. Part 2: Photographs.
Part 1 of this survey comprised a description of the luminometers (J. Biolumin. Chemilumin. 7, 77-108 (1992)) and was compiled in January and February 1992 from information available in public domain literature requested by and supplied to the author by numerous companies in the previous two months. This part includes photographs of around 40 of these instruments. It is intended that updates to this review will be published at least annually in this journal and suppliers are invited to provide full technical details of new luminometric equipment to the author.
Autoanalysis↗
Continuous fractionation of human plasma.
Continuous processing has been applied to human plasma fractionation by the cold ethanol process. On-line pH control of +/- 0.05 pH units, flow control of +/- 1%, and temperature control of +/- 0.5 degree C have been achieved. Optimization of precipitation pHs has been carried out for purifying plasma protein fractions and albumin. During precipitation, the irreversible nature of the pH overshoots has been demonstrated. Compared to the batch processing mode, the continuous scheme produces an increased yield between 6 to 11%.
Autoanalysis↗
Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system.
Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid), a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibrozil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.
Autoanalysis↗
Disopyramide analysis using REMEDi: comparison with EMIT and conventional high performance liquid chromatographic methods.
REMEDi (Rapid EMErgency Drug identification; Bio-Rad) is an automated high performance liquid chromatographic (HPLC) system designed to detect, identify and measure a range of basic and neutral drugs in 0.5-1.0 mL of urine or plasma/serum. We have evaluated REMEDi in the analysis of the antiarrhythmic drug disopyramide in patient samples. The specimens were also analysed by a conventional HPLC method, based on solvent extraction and UV detection (254 nm), and by EMIT. There were good correlations between the results obtained with each method (r = 0.91 or greater). REMEDi gave a lower mean result than EMIT [means +/- SD (mg/L): REMEDi 2.64 +/- 1.10, EMIT 3.14 +/- 1.51; t = 4.0, p less than 0.01; n = 25], but there were no other significant differences in mean results. The principal disopyramide metabolite, mono-N-desalkyldisopyramide, did not interfere in any method. Clearly REMEDi can be used for therapeutic drug monitoring of disopyramide provided enough sample is available.
Autoanalysis↗
An automated method for the determination of a new potential antiepileptic agent (CGP 33101) in human plasma using high performance liquid chromatography.
An automated analytical method utilizing laboratory robotics has been developed and validated for quantifying concentrations of a new antiepileptic drug candidate (CGP 33101) in human plasma. The robotic system aliquots the biological sample, adds the internal standard (CGP 23901) and pH 12 buffer, extracts the compounds from the basified matrix into an organic phase (methyl-t-butyl ether:dichloromethane, 2:1) and concentrates the extracts for reversed-phase, high performance liquid chromatographic (HPLC) analysis. The robotic system is directly interfaced with the HPLC system. Separation is achieved on a Hypersil 3 microns C18 column (4.6 x 50 mm) with ultraviolet detection of the analytes at 230 nm. Specificity was demonstrated by the lack of interfering peaks at the retention times for both the drug and internal standard. Recovery and reproducibility assessments indicated good accuracy (overall mean relative recovery of 102.7%) and precision (coefficient of variation of 4.4 to 7.7%) for CGP 33101 over the concentration range of 50-4000 ng/mL. The limit of quantification (LOQ) is 50 ng/mL. The method has been successfully applied to a clinical study in which normal volunteers received single oral doses of 400-1200 mg of this new drug candidate.
Anticonvulsants↗
Polypeptide sequencing by liquid chromatography mass spectrometry.
Key steps in a proposed automated system for polypeptide sequencing utilizing a liquid chromatograph mass spectrometer computer system have been tested with mixtures containing up to six model oligopeptides. At the low nanomole level it was possible to obtain complete sequence information for all components in many, but not all, of the mixtures tried. Interpretation of the results is complicated by the presence of numerous side-products formed in the derivatization process. Minimization of such impurities will be necessary to reduce the ambiguity of the sequence information resulting from more complex mixtures, such as those expected from the degradation of larger polypeptides, and to reduce sample requirements to the subnanomole level. However, the present system appears to have unique advantages over other proposed automated methods.
Amino Acid Sequence↗
Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources.
Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
Animals↗
Biomedical and clinical applications of automated single cell electrophoresis.
The automated single cell electrophoresis microscope Parmoquant has been used for the discrimination of lymphocytes and the study of the interaction of substances with cells and synthetic particles. Electrophoretic histograms allowed the determination of changes in the proportion of lymphocyte populations after kidney transplantation, during dialysis treatment, open heart surgery and during pregnancy. Discrimination of leukemic cells on the basis of electrophoresis was used as an additional parameter in diagnosis. In a mouse tumor model, histogram determination enabled the in vivo effect of the tumor necrosis factor on immune cells to be evaluated. Cell electrophoresis was shown to be suitable to detect the influence of antibodies, lectins and bacteria on the cell surface. Protein adsorption was studied on synthetic particles using cell electrophoresis. This method was applied to investigate the phenomena of blood interaction with biomaterials used in artificial organs and to determine differences in the protein composition of serum or other body fluids connected with diseases.
Animals↗
Automated amplification and sequencing of human mitochondrial DNA.
Part of the human mitochondrial D-loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M13-21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single-stranded DNA was generated. This was then sequenced directly by the diodeoxy chain termination method using dye-labelled universal sequencing primers in conjunction with a fluorescence-based DNA sequencer. This enabled a 403-base-pair hypervariable segment of the D-loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.
Amino Acid Sequence↗
Stability of capillary gels for automated sequencing of DNA.
Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)
Autoanalysis↗
cDNA sequence of three cysteine-rich clusters in the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer.
Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.
Amino Acid Sequence↗
Pulsed field sequencing gel electrophoresis.
The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.
Autoanalysis↗
Movement of fluorescence pattern after photobleaching: an accelerated procedure for DNA electrophoretic mobility analysis.
A new approach which is compatible with many of the existing procedures for the analysis of DNA species in gel electrophoresis is being demonstrated. It takes advantage of fluorescence photobleaching in order to create a sharp boundary between the stained and the (partially) photobleached DNA. By arbitrarily creating a stained DNA band of narrower width, the sensitivity to detect (averaged) DNA band movements has been increased. This feature permits measurements of time-dependent electrophoretic mobility over very short time periods. The approach can be used to shorten the running time of gel electrophoresis experiment and to increase the resolution because of the sharper boundary and narrower band width. With faster running time, diffusion of both DNA and dye in the gel also becomes less serious. Movement of fluorescence pattern after photobleaching also permits measurements of localized motions when the gel pores are small in comparison with DNA sizes. Experiments demonstrating some aspects of the proposed technique, as well as the anticipated limitations, are presented and discussed.
Autoanalysis↗
Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer.
Although automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10(-17) mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence-collecting yield. The emission and photodestruction cross-sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross-sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence-collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo-destructive, the cross-section of the destruction being about 3.8 x 10(-20) cm2 in buffer solution while that of Texas Red is 1.5 x 10(-21) cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 mW, with an Ar laser (488 nm) for FITC-DNA, and 18 mW, with an He-Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence-collecting efficiency by a factor of 4, and an He-Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra-high sensitivity of 5 x 10(-20) mole/band (S/N-4) was achieved under optimized conditions.
Autoanalysis↗
[Use of the combined Kjeldahl-wet digestion/Berthelot-reaction for determining nitrogen in biological materials. 2. Design and testing of a partially automated apparatus for routine determination of nitrogen].
The authors describe the design and the mode of operation of a partially automated analyzer system, based on the combination of the Kjeldahl wet digestion with the Berthelot reaction, for the determination of nitrogen in biological materials. The results from three years of operation are discussed from the view-points of variability of the materials to be tested, of accuracy and of precision. This analytical technique (with an accuracy of 98% and a precision of 4%) is suited for determining amino and amido nitrogen as well as non-aromatically bound ring nitrogen. The statistical evaluation of the analytical results revealed that the deviation of the measured values is mainly caused by the high-temperature digestion.
Amides↗