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The serology of Pseudomonas aeruginosa analysed by means of quantitative immunoelectrophoretic methods. III. Reproducibility of a polyvalent P. aeruginosa reference standard-antigen.

The reproducibility of a polyvalent Pseudomonas aeruginosa antigen (St-Ag) composed of a mixture of antigens from 4 O groups of this bacterium has been studied. Ten batches of St-Ag were produced, and each of these and each of the 10 batches of antigens from the 4 strains of P. aeruginosa were compared with St-Ag batch 1 by means of quantitative immunoelectrophoretic methods and a polyvalent antiserum (St-Ab) raised against St-Ag. Fifty-three of the 55 antigens of St-Ag were stable and could be reproduced with reasonable precision in all 10 batches, and 3 of the 4 strains of P. aeruginosa were stable in antigen composition in all batches. One of the strains (0-5A) had lost 2 antigens in the last 5 batches, and the concentrations of 7 other antigens were simultaneously changed, reflecting a smooth-rough dissociation. The disappearance of the 2 antigens in the latest 5 batches of 0-5A was also reflected in similar changes in the antigen composition of the latest 5 batches of St-Ag.

Antigens, Bacterial↗

Evaluation of the E test by using selected gram-positive bacteria.

The E test (AB Biodisk NA Inc.) was compared with standard reference methods using the National Committee for Clinical Laboratory Standards's recommendations for determining the MICs of four selected antibiotics against 208 clinical isolates of gram-positive bacteria. These bacteria included 32 strains of Streptococcus pneumoniae, 25 strains of Enterococcus faecium, 20 strains of oxacillin-sensitive Staphylococcus aureus (OSSA), 96 strains of oxacillin-resistant S. aureus (ORSA), and 35 strains of coagulase-negative staphylococci. Evaluation included MIC accuracy within 1 dilution, reproducibility testing, and cost analysis. There was 94% agreement between the E test and the reference method in testing S. pneumoniae and penicillin G. There was 92% agreement with ampicillin and 100% agreement with vancomycin in testing E. faecium isolates. Accuracy of the oxacillin E test with staphylococci was significantly improved by the use of salt-supplemented Mueller-Hinton agar, for an agreement of 100% with coagulase-negative staphylococci and oxacillin-sensitive S. aureus and that of 85% with oxacillin-resistant S. aureus, with no major discrepancies. The E test with American Type Culture Collection isolates and clinical strains gave excellent reproducibility and was less costly than microdilution panels when used to test fewer than three antibiotics. The E test is a simple, reliable, reproducible, and cost-effective method for MIC determination for gram-positive organisms.

Enterococcus faecalis↗

Determination of metals in composite diet samples by inductively coupled plasma-mass spectrometry.

A study was conducted to evaluate the applicability of inductively coupled plasma-mass spectrometry (ICP-MS) techniques for determination of metals in composite diets. Aluminum, cadmium, chromium, copper, lead, manganese, nickel, vanadium, and zinc were determined by this method. Atmospheric pressure microwave digestion was used to solubilize analytes in homogenized composite diet samples, and this procedure was followed by ICP-MS analysis. Recovery of certified elements from standard reference materials ranged from 92 to 119% with relative standard deviations (RSDs) of 0.4-1.9%. Recovery of elements from fortified composite diet samples ranged from 75 to 129% with RSDs of 0-11.3%. Limits of detection ranged from 1 to 1700 ng/g; high values were due to significant amounts of certain elements naturally present in composite diets. Results of this study demonstrate that low-resolution quadrupole-based ICP-MS provides precise and accurate measurements of the elements tested in composite diet samples.

Animals↗

Variability in the sensitivity of nine enzyme-linked immunosorbant assays (ELISAs) in the measurement of human interleukin 6.

ELISAs employing several combinations of polyclonal and monoclonal anti-human IL-6 antibodies for the detection of interleukin 6 (IL-6) in cell culture supernatants were developed and compared. They were found to have differing sensitivities in the measurement of standard preparations of recombinant human (rh) IL-6 (WHO reference standard) as well as conventional preparations of IL-6 produced by stimulated human peripheral blood mononuclear cells. Thus, an ELISA that was optimal for detecting rhIL-6 standard was suboptimal for detecting IL-6 in cell culture supernatants. The presence of soluble IL-6 receptor (sIL-6R) or the signal-transducing gp130 molecule or serum did not alter the levels of IL-6 detected by these ELISAs. Therefore, the observed variability in sensitivity of the assays may be due to differences in epitope specificity of the various monoclonal antibodies used and the heterogeneity of IL-6 secreted into culture supernatants.

Antibody Specificity↗

A method for correcting for the variability of inhibitory effects of soluble human interleukin 1 receptor II measured by different ELISAS.

Seven ELISAs were developed by using several combinations of anti-human IL-1beta antibodies for detecting interleukin 1beta (IL-1beta) in cell culture supernatants. These ELISAs have different sensitivities in detecting standard preparations of recombinant human IL-1beta (WHO reference standard) compared with conventional preparations of IL-1beta produced by stimulated human peripheral blood mononuclear cells. The observed differences were attributed to differences in epitope specificity of the various monoclonal antibodies used and the heterogeneity of IL-1beta secreted into culture supernatants. The presence of soluble IL-1 receptor type I did not alter the levels of IL-1beta detected by these ELISAs. However, soluble IL-1 receptor type II interfered with the detection of IL-1beta to different degrees in these ELISAs. A method involving standarization by means of separate measurement of the amount of receptor and its inhibitory effect in the IL-1beta ELISA, yields consistent estimates of the correct IL-1beta levels.

Artifacts↗

Comparison of the Murex Candida albicans CA50 test with germ tube production for identification of C. albicans.

A total of 502 yeast isolates were tested with the 30-min MUREX Candida albicans CA50 (Norcross, GA) test for presumptive identification of C. albicans. The results were compared with the standard 2-h germ tube test, which was the reference standard. Of the 502 isolates, 316 were C. albicans and 186 were non-C. albicans. Identifications were based on germ tube reactions; the API20C and chlamydospore agars were used when discrepant results persisted between the germ tube and MUREX test after repeat testing of the MUREX method. A total of 16 C. albicans gave negative results on initial testing with the MUREX test but were interpreted as positive when repeated. Three germ tube negative yeasts initially tested positive with the MUREX but were negative when repeated. Two additional yeast isolates gave incorrect results with the MUREX, even with repeat testing: C. albicans and C. lusitaniae. The initial sensitivity and specificity for the MUREX C. albicans CA50 test were 94.6% and 97.8%, respectively. As an addition to the study, two fetal bovine sera were compared for production of germ tubes; fetal bovine serum and Fetal Clone II. The testing found them to be in 100% agreement.

Aminopeptidases↗

[Study of reference material for excitation spectrum wavelength calibration of fluorescence spectrophotometer].

A reference material used for wavelength calibration of fluorescence spectrophotometer was found. The holmium doped oxide reference material GBW(E) 130112 is a kind of standard reference material for absorption spectrophotometer. It can emit 547.7 nm fluorescence when excited by xenon lamp light. The excitation spectrum of 547.7 nm fluorescence was measured. It was found that the measured peaks of excitation spectrum are positioned at 333.56, 360.43 and 418.39 nm, respectively, which are coincident with the true values 333.8, 360.9 and 418.5 nm of reference material certification. It was illustrated that the holmium doped oxide reference material GBW(E)130112 could be used as reference material for the excitation wavelength calibration of the fluorescence spectrophotometer. Its property could be enhanced very much if high luminescent efficiency material is selected as rare earth ion doped matrix, and the purity is enhanced to reduce the cross relaxation.

Algorithms↗

Physical and chemical characteristics of UICC reference samples.

New data on the physical and chemical characteristics of the UICC standard reference samples are presented. Aspects such as fibre length and diameter distribution, composition and special electrical and magnetic properties are covered. The possibility of preparing samples with graded characteristics is raised. A new edition of the data sheets is proposed.

Asbestos↗

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Azo Compounds↗

Comparative evaluation of two methods to assay thyroglobulin serum concentrations in patients with differentiated thyroid carcinomas.

AIM: This study was aimed at the comparative assessment of the analytical and clinical performances of 2 tests for thyroglobulin (Tg) assays: the Dynotest Tg-Plus immunoradiometric assay (IRMA), a new method that might be of interest for its claimed superior sensitivity compared to other methods, and the HTGK-2 IRMA, one of the test currently used in clinical routine. METHODS: The study was performed in serum samples from 157 patients with differentiated thyroid carcinoma (DTC). The clinical sensitivity of the test was evaluated in patients with and without thyroid stimulating hormone (TSH) suppression. RESULTS: The lowest detectable Tg concentration values and the within-assay coefficient of variation (CV) were 0.4 and 0.8 microg/L and 5% and 3% for the Dynotest Tg-Plus assay and the HTGK-2 assay, respectively; the between-assay CV was 6% for both assays. The clinical results of the Dynotest Tg-Plus and those of the HTGK-2 kit were similar in both DTC patient populations, either under or off the TSH suppressive treatment. In spite of the manufacturer's statement that the calibrators of both assays had been standardized against the same common reference (standard CRM 457 of the Community Bureau of References), the Dynotest Tg-Plus test underrated by a factor of 0.5 the Tg values measured by means of HTGK-2 IRMA. CONCLUSION: The sensitivity of the Dynotest Tg-Plus IRMA appears to be similar to that of the HTGK-2 assay.

Adolescent↗

Reference values and standards for paired organs.

When estimating reference values for measurements on paired organs, the standard deviation for a single measurement can be obtained to a good approximation simply by ignoring the pairing. In practice, however, there are advantages in the use of conventional standards for pair means and differences.

Anatomy↗

[Purification and fingerprinting development of Salvia miltiorrhiza Bunge by high-speed counter-current chromatography].

In an attempt to apply high-speed counter-current chromatography HSCCC for TCM fingerprints, the separation and purification of the Chinese medicinal plant Salvia miltiorrhiza Bunge of different localities was realized using the technique. The equipments used include a HSCCC (TBE-300) of Shenzhen Tauto Biotech containing three connected preparative coils (diameter of tube = 2.6mm, total volume = 300mL) and a 20mL sample loop and a HPLC from Shimadzu of Japan with a Ultrasphere C18 column (150 x 4.6mm ID, 5microm) and a 20microL sample loop. Salvia miltiorrhiza Bunge samples from 3 locations were separated by HSCCC in a Step-wise elution program with solvent systems A (hexane:ethanol: water = 10:5.5:4.5) and B (hexane:ethanol: water = 10:7:3) at a speed of 900 r/min and a flow-rate of 2mL/min. All the 12 peak fractions were eluted within 13 hours. The contents of each component varied greatly in different samples, which confirmed previous observation that the locations and climates have a great impact on the TCM quality and also indicated a quality control system is necessary to safeguard the quality of the herb. The retention times of the 12 peak fractions from crude extracts of the samples were collected by HPLC and the absorption spectrums of the corresponding peaks were identified. The 12 components of the three crude samples were readily distinguishable and can be used as fingerprints of S. miltiorrhiza Bunge. The relative standard deviation of the HSCCC retention times was less than 3%, which satisfies the requirement of the national standard reference index. The components 7, 8 and 11 from the standards were identified to be crypototanshinone, tanshinone I and tanshinone II A respectively. This study demonstrates that if it is possible to apply HSCCC for TCM fingerprinting, especially with samples of high viscosity and highly absorptive components. The precision and the run time of fingerprinting can be further improved if larger volume and a temperature control system is used. With these and other improvements, HSCCC is expected to play an important role in TCM development.

Abietanes↗