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PubMed1999

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA, an automated coagulation analyzer.

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p < 0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p < 0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.

Automation↗
PubMed1996

Factors in standardizing automated cholinesterase assays.

A scientific panel assembled by the U.S. Environmental Protection Agency (EPA) determined that variability in cholinesterase (ChE) activities in the agency's pesticide/animal study database likely was due to a lack of accepted guidelines for ChE methodology. A series of trials was held in which participating laboratories measured ChE activity in blood and brain samples from untreated and pesticide-treated rats using a colorimetric assay method. The degree of inhibition of ChE activity in plasma and brain samples compared to controls was consistent among most of the laboratories. The ChE activity in erythrocyte samples differed more between laboratories due to a high blank, low erythrocyte AChE activity and hemoglobin absorption at the wavelength of the assay. Strategies are suggested for minimizing the variability of ChE activity in hemoglobin-rich samples.

Acetylcholinesterase↗
PubMed1988

Preliminary characterization of extracellular proteolytic enzymes of dermatophytes by chromogenic substrates.

Thirty-eight chromogenic substrates were used to study the specificity of the proteolytic enzymes of seven species of dermatophytes and three related keratinolytic soil fungi. The source of enzymes were cultivation fluids from cultures of the fungi grown on human hair. The overall specificity of the enzymes of all the keratinolytic fungi was very similar. Aminoacyl- and dipeptidyl-4-nitroanilides, substrates of aminopeptidases and dipeptidyl aminopeptidases respectively, were poor substrates compared to aminoterminally blocked oligopeptidyl derivatives. Of the latter, the best substrates were those with phenylalanine, leucine, alanine, methionine or arginine (i.e. amino acids with hydrophobic or basic side chains) in the P1 position. Of the amino acids in the P2 position, proline was the most effective at accelerating the hydrolysis of the respective substrates. Positions P3 and P4 and even the aminoterminal protecting group were also of importance. The specificity profiles of the proteolytic enzymes corresponded best to those of some well characterized serine proteinases (chymotrypsin, elastase).

Arthrodermataceae↗
PubMed2006

TOP2A and HER2 gene amplification as predictors of response to anthracycline treatment in breast cancer.

The purpose of this study was to evaluate amplification of topoisomerase IIalpha (TOP2A) and HER2 genes as predictors of response to chemotherapy in advanced breast cancer. Gene copy number of TOP2A and HER2 were analysed with chromogenic in situ hybridization (CISH) on paraffin-embedded tissue sections from the primary tumour of 85 patients treated with anthracycline containing chemotherapy. TOP2A gene amplification was present in 14 (16%) and HER2 gene amplification in 38 (45%) of the primary tumours. Two of the 14 cases with TOP2A amplification were amplified without concurrent HER2 amplification. Neither TOP2A nor HER2 gene amplification were significantly associated with response to chemotherapy (p = 0.35 and p = 0.49, respectively).

Adult↗
PubMed2003

ABC chromogenic agar: a cost-effective alternative to standard enteric media for Salmonella spp. isolation from routine stool samples.

Salmonellosis is the second most common cause of bacterial gastroenteritis, yet the yield from routine stool culture is low. Commonly used selective enteric media have poor specificities for salmonella identification, resulting in a high laboratory workload. A special chromogenic medium, ABC agar, is a promising alternative but its cost-effectiveness has not been evaluated in diagnostic laboratories. A collaborative study is therefore undertaken in two district general hospitals laboratories. Routine stool samples (n=866) were subcultured onto ABC agar half plates after selective enrichment in selenite broth. Similarly, 246 and 620 stool samples were subcultured onto desoxycholate lactose sucrose (DCLS) and xylose lactose desoxycholate (XLD) whole agar plates, respectively. Salmonella spp. were isolated from only 14 (1.6%) of stool samples tested. Specificity was significantly higher for ABC (98%) than DCLS (67%) or XLD (78%) agars. Welcan workload units (ABC 4.8, XLD and DCLS both 7.3) and costs per specimen (ABC 1.26 pounds sterling, DCLS 3.81 pounds sterling and XLD 1.83 pounds sterling) were similarly lower with ABC agar. The results indicate that ABC chromogenic agar offers improvements in specificity, workload and costs over conventional enteric media for Salmonella spp. isolation.

Agar↗
PubMed2005

Evaluation of three commercial agar preparations for the presumptive identification of significant urinary isolates.

Three commercially available pre-poured chromogenic preparations--chromogenic urinary tract infection (UTI) medium (chromogenic UTI, Oxoid), CHROMagar Orientation (Becton Dickinson) and CPS ID2 (bioMérieux)--are evaluated in comparison to routine urine microbiology using cysteine lactose electrolyte-deficient (CLED) medium and conventional methods of identification and susceptibility testing by Vitek 1 for the majority of isolates. Most isolates were Escherichia coli, and a chromogenic medium has been shown to be a reliable, rapid and more economic medium on which to presumptively identify these organisms due to the substrates the strain utilises in the plate and the chromogen subsequently produced. However, the opacity of chromogenic UTI made the medium difficult to inoculate and read, although the colours were clear and strong. Although there was no statistical difference between CHROMagar Orientation and CPS ID2, the colours observed on the former were stronger. This meant that colony counting was possible at significant concentrations of 10(4) and 10(5) colony-forming units (cfu)/mL and it may be easier to detect mixtures that would indicate contamination. Chromogenic media are richer than CLED and a number of Lactobacillus spp. (normally regarded as normal flora) grew on this medium. These were not considered to be significant.

Bacteria↗
PubMed2005

Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method.

BACKGROUND: Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria. METHODS: Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate. RESULTS: The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum. CONCLUSIONS: The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones.

Antioxidants↗
PubMed2006

Usefulness of Candida ID2 agar for the presumptive identification of Candida dubliniensis.

CHROMagar Candida and Candida ID2 are widely used for the isolation and presumptive identification of Candida spp. based on the color of the colonies on these two media. We have studied the usefulness of these chromogenic media for differentiating Candida dubliniensis from Candida albicans isolates. One hundred isolates of C. dubliniensis and 100 C. albicans isolates were tested on Candida ID2, CHROMagar Candida (CHROMagar), and CHROMagar Candida reformulated by BBL. CHROMagar Candida and CHROMagar Candida BBL did not allow a clear differentiation of the two species based upon the shade of the green color of C. dubliniensis colonies. However, on Candida ID2, all C. dubliniensis isolates produced turquoise blue colonies whereas 91% of C. albicans colonies were cobalt blue. The sensitivity and the specificity for differentiating between C. dubliniensis fromC. albicans on Candida ID2 were 100% and 91%, respectively; whereas on CHROMagar Candida these values were 63% and 89% and on CHROMagar Candida BBL they were 18% and 98%. Candida ID2 agar provides a simple and accurate laboratory approach for the identification and differentiation of C. dubliniensis on the basis of the colony color.

Agar↗
PubMed1981

3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.

The use of 3,3',5,5' - tetramethylbenzidine as non-mutagenic chromogen for the end point determination in enzyme-immunoassay (EIA) is described. In sandwich EIAs for HCG and HBsAg and in a competitive EIA for testosterone, the colour yield with TMB was superior to that obtained with o-phenylene diamine (OPD), which was by far the best chromogen for horse-radish peroxidase until now. This led to an improvement of sensitivity and precision of the assays and makes EIA even more competitive with other types of immunoassays.

Animals↗
PubMed1995

Use of progesterone-3(O-carboxymethyl oxime)-horseradish peroxidase in a sensitive microtitre-plate EIA and its application to a visual membrane EIA of progesterone.

A simple method of visual membrane enzyme-immunoassay (EIA) for the detection of progesterone is described. When two types of progesterone-horseradish peroxidase (HRP) tracers were challenged for binding, in the presence of progesterone, to the monoclonal anti-progesterone antibody, 15A, coated on the microtitre plate, the HRP conjugated at the C-3 position (A-ring) of progesterone competed more effectively with progesterone to the binding site of the monoclonal antibody (mAb) than HRP conjugated at the C-11 position of the C-ring. By using this combination of mAb, 15A, and progesterone-3(O-carboxymethyloxime)-HRP (P-3CMO-HRP), we developed a visual membrane EIA system in which free progesterone in the sample could be quantified by the degree of color development. In this system, free progesterone competed with P-3CMO-HRP for binding sites of mAb immobilized on the nitrocellulose membrane. The stable grey color was formed on the surface of membrane for progesterone-negative and no color for progesterone-positive sample using 3,3'-diaminobenzidine (DAB) with Co2+ as an insoluble substrate solution. To examine whether tetramethylbenzidine (TMB) can substitute for DAB in membrane EIA, an experiment was conducted where TMB was used as an insoluble substrate.

3,3'-Diaminobenzidine↗
PubMed1995

Non-instrumental immunoassay based on coloured polyacrolein latex: application to group-specific polysaccharide of Streptococcus pyogenes.

Non-instrumental immunoassay methods based on immunofiltration and microtiter particle agglutination (MPA) techniques have been developed using coloured polyacrolein latex. These methods have been applied to the quantification of the group-specific polysaccharide, A-PS, of S.pyogenes (group A Streptococcus) and compared to the standard ELISA tests. The assay with the ability to detect the lowest concentration of antigen was MPA; as little as 0.05 ng A-PS/ml or 10(4) cells/ml could be detected in 1.5 h. In comparison to ELISA test the sensitivity of MPA was 10 times higher and the procedure of the assay was much more simple. The sensitivity of the immunofiltration assay using both enzyme and latex markers was shown to be the same (50 ng A-PS/ml) and the duration of the assay 3-5 min. No cross-reactions of latex conjugates with non A Streptococcus cell lysates have been observed. The developed methods are easy to perform and require neither sophisticated equipment nor specially trained personal.

Acrolein↗
PubMed1997

Non starch polysaccharide hydrolyzing enzymes as feed additives: detection of enzyme activities and problems encountered with quantitative determination in complex samples.

Chromogenic substrates, an agar diffusion assay and viscosity reduction were used to estimate beta-glucanase and xylanase activities in water soluble extracts of different feedstuffs and digesta supernatants. The dinitrosalicylic acid reducing sugar method was employed to calibrate results from different methods based on international units (IU, glucose equivalents). The detection of dye release from chromogenic substrates was a suitable method, allowing the detection of 0.05 IU of enzyme activity per ml of extract, although measurements in digesta supernatants were limited in linearity (0.1-0.5 IU/ml supernatant). With the agar diffusion assay the detection of enzyme activity was possible over a wider concentration range (extracts: 0.05-1 IU/ml, digesta supernatants: 0.1-1 IU/ml), but visual evaluation led to inaccurate measurement. Accuracy can be improved by computer based evaluation of digital images. The use of viscosity reduction produced linear standard curves from 0.01 to 0.5 IU/ml in feed extracts, but reliability of measurements depended on modification of substrates. Quantification of enzyme activities was influenced by matrix effects of complex samples. Cereal dependant differences were found in various extracts of feed mixtures and cereal extracts. Digesta supernatants partly inhibited enzyme activity, depending on the origin of the sample. Interaction of substrates with digesta components varied between methods. The sensitivity of the methods is comparable, however, all methods require specific calibrations to account for matrix- and enzyme specific effects.

Animal Feed↗
PubMedDate not supplied

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics.

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.

Biological Transport↗
PubMed2001

Yeast identification in the clinical microbiology laboratory: phenotypical methods.

Emerging yeast pathogens are favoured by increasing numbers of immunocompromised patients and by certain current medical practices. These yeasts differ in their antifungal drug susceptibilities, and rapid species identification is imperative. A large variety of methods have been developed with the aim of facilitating rapid, accurate yeast identification. Significant recent commercial introductions have included species-specific direct enzymatic colour tests, differential chromogenic isolation plates, direct immunological tests, and enhanced manual and automated biochemical and enzymatic panels. Chromogenic isolation media demonstrate better detection rates of yeasts in mixed cultures than traditional media, and allow the direct identification of Candida albicans by means of colony colour. Comparative evaluation of rapid methods for C. albicans identification, including the germ tube test, shows that chromogenic media may be economically advantageous. Accurate tests for single species include the Bichrolatex Albicans and Krusei Color tests, both immunologically based, as well as the Remel Rapid Trehalose Assimilation Broth for C. glabrata. Among broad-spectrum tests, the RapID Yeast Plus system gives same-day identification of clinical yeasts, but performance depends on inoculum density and geographic isolate source. The API 20 C AUX system is considered a reference method, but newer systems such as Auxacolor and Fungichrom are as accurate and are more convenient. Among automated systems, the ID 32 C strip, the Vitek Yeast Biochemical Card and the Vitek 2 ID-YST system correctly identify >93% of common yeasts, but the ID-YST is the most accurate with uncommon yeasts, including C. dubliniensis. Spectroscopic methods such as Fourier transformed-infrared spectroscopy offer potential advantages for the future. Overall, the advantages of rapid yeast identification methods include relative simplicity and low cost. For all rapid methods, meticulous, standardized multicenter comparisons are needed before tests are fully accepted.

Candida↗
PubMed1995

The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae.

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.

Caveolin 1↗