Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera.
Kullertz G. Luthe S. Fischer G.
Max-Planck Research Unit, Enzymology of Protein Folding, Halle/Saale, Germany.
An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann-Whitney rank-sum test P < 0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 degrees C was stable for at least 20 h.
Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system.
Bossuyt X. Schiettekatte G. Bogaerts A. Blanckaert N.
Department of Clinical Pathology, University Hospital of Leuven, Belgium. firstname.lastname@example.org
We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.
Comparison of the Axis %CDT TIA and the CDTect method as laboratory tests of alcohol abuse.
Viitala K. Lahdesmaki K. Niemela O.
EP Central Hospital Laboratory, Seinajoki, Finland.
Carbohydrate-deficient transferrin (CDT) has been suggested as a specific marker of alcohol abuse. We designed this study to compare the conventional CDTect method (Pharmacia & Upjohn) and the new semiautomated Axis %CDT turbidimetric immunoassay (%CDT TIA) for their diagnostic performance to identify problem drinking. The sensitivities of the %CDT TIA and CDTect for correctly classifying heavy drinkers (n = 90) were 29% and 59% with the thresholds currently recommended by the manufacturers, respectively. In the control group (n = 114), which included hospitalized patients with abnormal serum transferrin concentrations, the CDTect assay gave 21 false-positive values (18%), whereas the %CDT TIA showed 100% specificity. With the cutoff limits based on the present healthy control group (mean + 2 SD), the sensitivities of the %CDT TIA and CDTect were 61% and 86%, respectively. For men, the ROC plot area of the CDTect results in comparisons of alcohol abusers and healthy controls was significantly (P < 0.05) higher than that of the %CDT TIA results, whereas for women, there was no significant difference in this respect. The slope and intercept (with 95% confidence intervals) for linear regression between CDTect and %CDT TIA were 0.13 (0.12-0.15) and 1.16 (0.73-1.59), respectively (S(y/x) = 1.51, r = 0.744). CDTect results correlated positively with serum transferrin (r = 0.224, P < 0.001), whereas the %CDT TIA results showed a slight inverse correlation with serum transferrin (r = -0.132, P = 0.07). The data suggest that CDTect is more sensitive than %CDT TIA in detecting drinking problems. However, the %CDT TIA method yields more specificity when analyzing samples from patients with high serum transferrin concentrations.
Evaluation of a homogeneous assay for high-density lipoprotein cholesterol: limitations in patients with cardiovascular, renal, and hepatic disorders.
Simo JM. Castellano I. Ferre N. Joven J. Camps J.
Centre de Recerca Biomedica, Hospital Universitari de Sant Joan/carrer, Catalunya, Spain.
We evaluated the performance of a homogeneous assay for the automated measurement of high-density lipoprotein cholesterol (HDL-C) and compared it with a conventional precipitation technique in the following groups of people: control subjects (group A), clinically-healthy elderly (group B), myocardial infarction patients (group C), nephrotic syndrome patients (group D), and liver cirrhosis patients (group E). The performance of the technique was acceptable with respect to precision, accuracy, linearity, and detection limit. Triglycerides up to 40 mmol/L and bilirubin up to 150 micromol/L did not cause interferences. Hemoglobin decreased HDL-C measurements. Samples were stable at -20 degrees C for up to four months. Bland-Altman plots showed a good agreement between both techniques in the control group but with a progressive divergence in the patient groups B to E. Results indicate limitations of the technique in certain clinical conditions and, coincidentally, the need for reliable calibration materials.
Optimized serum pancreolauryl test for differentiating patients with and without chronic pancreatitis.
Dominguez-Munoz JE. Malfertheiner P.
Department of Gastroenterology, University of Magdeburg, Germany. email@example.com
The serum pancreolauryl test has limited sensitivity for detecting mild pancreatic insufficiency. The aim of this study was to optimize the serum pancreolauryl test so as to increase the probability of positive results in patients with chronic pancreatitis. The study had three parts. First, the sampling time was optimized by analyzing retrospectively the frequency of fluorescein peaks at different times from 0 to 240 min in 560 consecutive patients. Second, the calculation of serum fluorescein concentrations by means of a standard calibration factor was prospectively compared in 271 consecutive patients with a modification involving a specimen-specific calibration factor for each patient. Third, the clinical utility of the intravenous injection of secretin before ingestion of the test meal was prospectively evaluated in a further 32 patients. As a result, the optimized serum pancreolauryl test developed differs from the former version of the test in utilizing intravenous administration of secretin before the test meal, calculation of serum fluorescein based on specimen-specific calibration factors, and blood samples taken only at 0 (basal), 120, 150, 180, and 240 min. This optimized pancreolauryl test was abnormal more frequently in patients with chronic pancreatitis than was the formerly used test, especially for cases of mild and moderate disease.
Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene.
Gelfi C. Perego M. Righetti PG. Cainarca S. Firpo S. Ferrari M. Cremonesi L.
Instituto Di Technologie Biomedicrye, Consiglio Nazionale delle Ricerche, Milan, Italy.
The poly-T tract in intron 8 of the cystic fibrosis conductance transmembrane regulator (CFTR) gene exists in three variants, 5T, 7T, and 9T. The 7T and 9T variants generate a predominantly normal transcript, whereas the 5T variant engenders an anomalous product. The analysis of the poly-T tract is assuming increasing relevance, both to assess the implication of the CFTR gene in congenital bilateral absence of the vas deferens and to evaluate genotype-phenotype correlation in cystic fibrosis. Mapping of the poly-T tract has been performed by cumbersome and time-consuming methodologies. Capillary zone electrophoresis, combined with laser-induced fluorescence detection, was introduced for a rapid separation of the poly-T tract amplified products. As separation buffer, we adopted 200 mmol/L histidine (pH = pI = 7.6), and the capillary was filled with 10% polyacrylamide, allowing separations in less than 10 min. Capillary zone electrophoresis results were in perfect agreement with dot-blot analysis.
A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping.
Whitcombe D. Brownie J. Gillard HL. McKechnie D. Theaker J. Newton CR. Little S.
Zeneca Diagnostics, Gadbrook Park, Northwich, Cheshire, UK. firstname.lastname@example.org
We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5'-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.