Centrifugal Fast Analyzer Research Articles

Robotic enzyme amplification: a comparison of some kinetic properties of bovine liver, Candida utilis and Proteus sp. glutamic dehydrogenases

NADP(H)-specific Bakers yeast glucose 6-phosphate dehydrogenase (BYG6PDH) was paired, in turn, with each of three different source glutamate dehydrogenases (GDHs): NAD(P)-specific bovine liver (BLGDH), NADP-specific Candida utilis (CUGDH) and NADP-specific Proteus sp. (PSGDH) to constitute three enzyme cycling systems; (i) BYG6PDH/BLGDH; (ii) BYG6PDH/CUGDH; and (iii) BYG6PDH/PSGDH. When incorporated into an enzymatic cycling/amplification system for NAD kinase and run on a centrifugal fast analyzer (CFA), the microbial source enzyme CUGDH gave rise to a seven-fold greater amplification rate [21.5 x 10(3) cycles-1 (cph)] relative to that realized (3 x 10(3) cph) using the BYG6PDH/BLGDH cycling pair previously reported. Either of these cycling systems can be used as a flexible and general-purpose module for robotic amplification and data collection of NADP(H) linked enzymes as a user's requirements dictate. Although the BYG6PDH/PSGDH cycling pair produced a respectable cycling rate (14.4 x 10(3) cph), for reasons discussed the PSGDH enzyme was not considered a suitable replacement for BLGDH in an NADP(H) cycling system.

Evaluation of a new chromogenic substrate assay for the measurement of the prothrombin time

The chromogenic prothrombin time assay Nycotest Chrom was evaluated and compared with another chromogenic assay (Chromoquick) and with the well-known coagulometric Thrombotest. The intra-assay variation of the test was satisfying both in the normal range (CV = 3.6%) and in the therapeutical range of oral anticoagulation (CV = 3.5%). These values were somewhat higher than those of the Chromoquick test (1.3% and 1.1%) and those of the Thrombotest (2.3% and 2.5%). The inter-assay variation coefficient amounted to 3.3% in the normal range and in the therapeutical oral anticoagulation range to 4.4%. The respective values for the Chromoquick test were somewhat lower (2.2% and 3.9%), those of the Thrombotest in the normal range were also lower (2.1%), and in the therapeutical range they were much higher (9.2%). The coefficients of correlation of the Nycotest Chrom test in the therapeutical range were highest when comparing them with those for the Chromoquick test (r = 0.97; p < 0.0001) and somewhat lower in comparison with the Thrombotest (r = 0.95; p < 0.0001). The results of the three methods paralleled also in the starting phase of oral anticoagulation treatment. Storage at 4 degrees C and at 25 degrees C showed good stability of the Nycotest Chrom reagent for up to 24h. We conclude that the Nycotest Chrom assay is well suited for the measurement with a centrifugal fast analyser, which is attractive on behalf of the usually large series of request for prothrombin time measurements when monitoring oral anticoagulation therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of urease-inhibiting activity in serum from children infected with Helicobacter pylori.

In order to provide a basis for obtaining further information concerning the host response to Helicobacter pylori urease, four assay methods for detecting urease-inhibiting activity in serum were examined. A quantitative assay, established in a COBAS BIO centrifugal fast analyzer and based on detection of the consumption of NADH by glutamate dehydrogenase stimulated by ammonia production, was considered most suitable for large-scale serological work. Serum samples from 63 children (aged 5 to 16 years), 28 of whom had seropositive H. pylori gastritis, were assayed. One of the serum samples in this latter group showed significant inhibitory activity. This serum sample was one of 13 in the seropositive group known to bind to urease antigen. It showed no inhibitory activity against Bacillus pasteurii or jack bean urease. Protein A binding and heat treatment indicated that the inhibitory activity was immunoglobulin G mediated. The patient from whom this sample was collected showed no distinctive features in his illness. The COBAS BIO analyzer-based urease inhibition assay provides a new tool for studying one aspect of the host response to H. pylori infection.

Adaptation of an enzymatic cycling assay for NADP(H) measurement to the COBAS-FARA centrifugal analyzer

NADP(H) measurements by enzymatic amplification are described in which the interface step between cycling (glucose-6-phosphate and glutamic dehydrogenases) and indicator (6-phosphogluconic dehydrogenase) enzymes has been reconfigured, permitting the entire operation to run as a continuous assay on a centrifugal fast analyzer. This is accomplished by using the sequential load feature of the analyzer and incorporating either sodium dodecyl sulfate (SDS) or SDS and hydrogen peroxide as kill reagents to replace the thermal step (destruction of cycle enzymes by boiling). The ability of SDS to render a cycle inoperative during the run time of the indicator enzyme depends on the inherent resistivity and absolute amount of its enzyme proteins to this surfactant. Criteria used to judge the efficacy of a potential kill reagent are based on the sample blank time-response curve and the cycle product recovery by the indicator enzyme. Various other enzyme cycling systems which can be fitted to the centrifugal fast analyzer are highlighted.

Evaluation of a new method for cholinesterase determination

We report the evaluation of a new commercially available assay system for determination of the catalytic activity of serum cholinesterase (EC 3.1.1.8) and application of the method to a centrifugal fast analyzer. Serum cholinesterase activity is determined at 30 degrees C using para-hydroxybenzoylcholine as substrate. This reaction is coupled to a second reaction using para-hydroxybenzoate hydroxylase (EC 1.14.13.2) as coupling enzyme. Enzyme activity is measured kinetically by monitoring the decrease in absorbance at 340 nm of NADPH in the second reaction. The procedure is precise and the results obtained from normal and pathological sera show good correlation with those obtained by the alternative procedures employing propionylthiocholine, butyrylthiocholine and benzoylcholine as substrates. The reference range for 700 healthy subjects was estimated to be 140-345 U/L (95% central range, determined non-parametrically), with significant difference between males and females (155-353 U/L for men and 134-323 U/L for women, p less than 0.001).

Procedure for toxicological screening by use of a centrifugal fast analyzer.

Journal Article Procedure for toxicological screening by use of a centrifugal fast analyzer. Get access M P Bosomworth M P Bosomworth Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 31, Issue 6, 1 June 1985, Pages 1084–1085, https://doi.org/10.1093/clinchem/31.6.1084 Published: 01 June 1985

Immunological and enzymatic studies of erythrocytic delta-aminolevulinate dehydratase. Comparison of results obtained in normal and lead-exposed subjects.

The delta-aminolevulinate dehydratase (ALA.D) quantitative assay on a centrifugal fast analyser showed that subjects whose blood-lead level varies between 30 and 75 micrograms/100 ml (1.5 to 3.75 microM/l) react to blood intoxication by synthesizing de novo an amount of enzyme correlating to blood-lead levels. At higher concentrations, the reactional synthesis occurs very rarely. These results suggest that enzyme is constitutive, but also inductible as soon as its substrate accumulates; this last ability may disappear at high blood-lead levels: a hypothesis is proposed thereafter.

C reactive protein concentrations in neonates: determination by a latex enhanced immunoassay.

A latex enhanced immunoassay on a centrifugal fast analyser was used to compare serum C reactive protein concentrations in maternal and neonatal blood. In the neonate the C reactive protein concentration at birth was less than 1.0 mg/l; the concentration rose slightly during the first two weeks of life. There was no correlation between C reactive protein concentrations in maternal and neonatal sera. No significant difference was found between the C reactive protein concentrations in blood obtained by either heel prick or venepuncture.

Evaluation of a new continuous colorimetric method for determination of serum pseudocholinesterase catalytic activity and its application to a centrifugal fast analyser.

We report the evaluation of a new commercially available assay system for the determination of serum pseudocholinesterase (EC 3.1.1.8) catalytic activity, and its application to a kinetic analyser. The assay is based on the colorimetric method of Okabe et al. (Clin. Chim. Acta 80, 87-94 (1977]: choline, liberated from benzoylcholine by pseudocholinesterase, is oxidized by choline-oxidase (EC 1.1.3.17) to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a coloured compound with maximal absorbance at 500 nm. The procedure not only has the advantage of being continuous, colorimetric and totally enzymatic but also appears to be precise (between-day analysis gives coefficient of variation between 3.5 and 5.6%) and accurate; the results obtained from normal and pathological sera show excellent correlation with those obtained by the alternative procedures employing propionylthiocholine, acetylthiocholine and butyrylthiocholine as substrates.

Reduction of analytical variance by using a discrete-time data-weighting filter to estimate abrupt changes in batch-type processes : Part 2. Applications

A well known characteristic of analytical methods in clinical laboratories is the presence of a significant between-batch variance component. This is confirmed by an analysis of variance of data obtained for the determination of inorganic phosphate in blood serum with a continuous flow system. Based on a model describing the process fluctuations, the proposed discrete-time digital filter algorithm estimates abrupt changes in the process values between two runs of samples. Subsequent correction of the control sera data for these estimates leads to a reduction of about 50% in the variance. Similar results were obtained for other analytical methods including a centrifugal fast analyzer, an automatic discrete system and a semi-automatic coulometric titration.

Use of a cresol red dye system to measure temperature on a centrifugal fast analyzer: A. Brigandi, L. Mullaly and D. Grisley, Jr. (Eastman Kodak Company, Rochester, NY 14650)

Use of a cresol red dye system to measure temperature on a centrifugal fast analyzer: A. Brigandi, L. Mullaly and D. Grisley, Jr. (Eastman Kodak Company, Rochester, NY 14650)

A semi-automated procedure for the assay of 23 enzymes from Drosophila melanogaster

A semi-automated procedure for the spectrophotometric assay of 23 enzymes from Drosophila melanogaster was developed to screen for correlated genetic effects on the activities of different enzymes. The procedure allows for the assay of 18 cytosolic and 5 mitochondrial enzymes from a mass homogenate of 100 male imagoes. Most of the assays were performed with the use of a computer-interfaced GeMSAEC centrifugal fast analyzer that allows the simultaneous performance of 16 assays and for automatic data processing. Estimation of the total measurement error (extraction plus assay repeat-ability) gives coefficients of variation less than 10% for most of the enzymes.

The application of a sensitive uricase-peroxidase coupled reaction to a centrifugal fast analyser for the determination of uric acid

A uricase-peroxidase coupled system, for the determination of uric acid, applied to a CentrifiChem-500 centrifugal fast analyser is described. Relatively large amounts of ascorbic acid, due to the inclusion of an ascorbate oxidase urine diluent, and hemoglobin appear not to interfere with the procedure while the incorporation of potassium ferrocyanide into the reagents has led to the near-total elimination of bilirubin interference. The incubation period is relatively short compared with other similar procedures and the one reagent system has made the procedure simple to perform. The use of sodium 2-hydroxy-3,5-dichlorobenzenesulfonate and 4-aminoantipyrene in the oxidative coupling reaction has incorporated the advantages of increased sensitivity, over phenol-4-aminoantipyrene systems, as well as the amenability of the reagent towards lyophilization or “dry-fill”.

Pig brain aldose reductase: A kinetic study using the centrifugal fast analyzer

1. 1. Initial velocity and product inhibition studies were performed on porcine brain aldose reductase (alditol:NADP + 1-oxidoreductase, EC 1.1.1.21) previously purified to apparent homogeneity. 2. 2. All rate studies were carried out on a centrifugal fast analyzer using d-xylose as substrate. 3. 3. The data, which indicate a sequential mechanism, are compatible with an ordered bi bi or an iso Theorell-Chance pattern of substrate addition-product release. 4. 4. Michaelis constants were K m xyl 4.1 ± 0.3 mM and K m NADPH 3.1 ± 0.2 μM. 5. 5. The advantages that accrue from the use of the centrifugal fast analyzer for reaction rate studies with enzymes (e.g. simultaneous multiple-reaction initiation and parallel monitoring) are pointed out.

Quantitation of human total IgG, kappa IgG and lambda IgG in serum using monoclonal antibodies

Monoclonal antibodies to human IgG have been applied, in parallel with polyclonal antisera, to the routine quantitation of human IgG. Two monoclonal antibodies directed against spatially distinct epitopes have been used in combination to form insoluble complexes exhibiting turbidity. Quantitation was performed using a centrifugal fast analyser and a correlation coefficient of 0.979 was obtained for the two estimations. The technique has been further developed to allow the separate quantitation of kappa IgG and lambda IgG in whole serum.

Erythrocyte transketolase, a new semi-automated method

This paper describes a method to determine transketolase activity in erythrocytes, with or without addition of thiamine-pyrophosphate, by quantitating the formation of glucose-6-phosphate formed during the incubation of hemolysed blood with ribose-5-phosphate. Glucose-6-phosphate is determined enzymatically, after deproteinisation of the sample, using a centrifugal fast analyser. The proposed method gave a correlation of r = 0.89 with an accepted colorimetric method. Reference values for blood are in the range 32–101 U/l.

Development and Improvement of a Commercial Uric Acid Enzymatic Determination Kit on a Centrifugal Fast Analyzer.

The performance of a uric acid determination kit has been evaluated for five months, under routine conditions, in a General Hospital Biochemical Laboratory. An anomalous increment in fresh serum blanks was noted in the kit when first introduced. This interference, probably due to alcohol dehydrogenase contamination, was corrected by addition of 50 mmol/l (NH4)2SO4 to the reagent. Results obtained with this modified reagent correlate perfectly with those obtained with modified kits by Smith Kline Instruments (SKI), and with many other determination methods. Correlations are discussed and explanations for differences in statistical data are offered. Within run and between run precision data are presented. The kit fits perfectly with the needs of centrifugal fast analyzers and discrete micro analyzers, on account of its speed, reliability and precision.

Detection of hepatitis B surface antigen with the miniature centrifugal fast analyzer. A modified reversed passive hemagglutination procedure.

A modified reversed passive hemagglutination test for the detection of hepatitis B surface antigen HBsAg is described. Sera and reagent cells coated with antibody to HBsAg (anti-HBs) are loaded separately into the rotor of a miniature centrifugal fast analyzer. The rotor is centrifuged briefly to transfer the components into its cuvettes. After mixing, the suspensions are allowed to stand at room temperature for 30 min, following which the rotor is again centrifuged and the absorbance of each cuvette is monitored. Cells suspended in serum containing HBsAg leave the light path more rapidly than cells suspended in sera free of antigen. The magnitude of change in absorbance varies directly with the concentration of the antigen. In 45 sera tested by the conventional V-plate technique, findings were as follows: 21 positive, 19 false positive and 5 negative. The automated procedure unequivocally differentiated the 21 positives; results for the false positive and negative specimens were identical and clearly distinguishable from the positive results. The automated procedure enhances specificity, offers equivalent sensitivity, and results that are quantitative and objective.

Nephelometric measurement of specific proteins using a centrifichem 400

A rapid precise assay for albumin and immunoglobulins has been developed using a centrifugal fast analyser. Samples are pre-diluted with saline. Appropriate high-titre antiserum is diluted with a polyethylene glycol (PEG)/sodium fluoride solution. This reagent is stable for 3 mth at 4 °C. Reaction times vary from a minimum 3.5min (IgG, albumin) to a maximum of 12min for IgA, IgM. Precision is considerably better than that obtained from radial immunodiffusion (RID), coefficients of variation being typically less than 3% within run, and less than 6% between runs. Cost of assays, based on reagents, is approximately ½ to ¼ that of RID, depending on choice of antisera used. The performance of a range of commercial antisera will be presented.

A kinetic enzymatic method for the urine and serum α-amylase determination with the centrifugal fast analyzer

A kinetic enzymatic method for the urine and serum α-amylase determination with the centrifugal fast analyzer