DIFFERENCE TASTE THRESHOLDS FOR SUCROSE IN WATER AND IN ORANGE JUICE: AN INTERLABORATORY STUDY

Difference taste thresholds, expressed as jnd values or Weberratios, were determined for sucrose in water and in orange juiceat laboratories in Sweden, U.S.A., Poland and Switzerland usinga method of constant stimuli. The following total arithmeticmean values of all 172 individual jnd values were obtained:0.266 and 0.400% sucrose at 2 and 5% sucrose in water, respectively;0.977 and 1.19% sucrose at 1.5 and 3.75% sucrose in orange juice,respectively. The frequency distributions of the individualvalues were asymetrical and showed a large variation among subjects.The results of some additional experiments at 2 and 5% sucrosein orange juice, performed only by the Polish laboratory, arereported also. Significance analyses performed according to one parametricmethod (t-test), using pooled data of groups of subjects, andone non-parametric method (Mann-Whitney's U-test), using individualthreshold values, gave the same conclusion in practically allcases. The data indicated that females had slightly lower average discriminationthresholds than males. There was a significant degree of correlationbetween subjects' discriminatory ability at different concentrationsof sucrose in each of the two media. Few significant differences between the laboratories were foundfor sucrose in water, whereas for sucrose in orange juice thefollowing rank order, from lowest to highest average jnd value,among the laboratories was obtained for both concentrationstested: Poland < U.S.A. < Sweden = Switzerland. Some speculationswere advanced as partial explanation for the differences amongthe laboratories. ^*Formerly Johansson     

猪口蹄疫病毒与猪肠道病毒在细胞培养中的基因重组——病毒重组及一个重组株的初步鉴定

利用两株不同理化和生物学特性的不同属小RNA病毒,即口蹄疫病毒(FMDV)与猪肠道病毒(EV),建立了病毒基因重组,克隆筛选和鉴定的方法,初步证实,在实验室条件下它们可以进行基因重组,本文为研究自然界病毒变异机理,不同属病毒的基因重组提供了依据。     

Comparability of poliovirus neutralizing antibody tests.

A serology panel was used to assess the comparability of results of poliovirus neutralizing antibody assays. Five laboratories from three countries provided results with seven in-house methods. A high degree of within-laboratory reproducibility was seen for all laboratories and methods, with 94% of results for repeat titrations within plus or minus one twofold dilution. Considerable differences in sensitivity between methods were, however, observed. The period of serum/virus incubation prior to inoculation of cells was shown by one laboratory to have a 10-fold effect on titres, and another laboratory showed that use of Sabin or wild-type challenge virus significantly influenced results for types 1 and 3. Expression of results as relative potencies abolished these difference due to method. The difference in sensitivity between laboratories for end-point titres (if one very sensitive method was excluded) was around fourfold for types 1 and 2, and ninefold for type 3. This range increased to 15-fold for type 2; and 20-fold for types 1 and 3 if the most sensitive method was included. Expression of results relative to a standard considerably improved agreement with a residual variation of around fourfold. Therefore, expression of results from serological studies, including vaccine trials, in International Units (i.e. relative to the new International Standard) will assure that comparisons between studies can be made with confidence.     

Intra- and interlaboratory performance of antibiotic disk-diffusion-susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments

In the course of an international research project on hazard analysis of antimicrobial resistance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquaculture environments was sent by the central laboratory to the other participating laboratories. In each laboratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were performed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of subcultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global averaging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indicated that the use of either ISA or MH medium in DD testing has a limited impact on the method's precision among different laboratories. In conclusion, the current study has provided a validated SOP to promote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive breakpoints should be specifically designed and validated for aquaculture drugs and organisms.     

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

An international collaborative study on foot and mouth disease virus assay methods. 1. Virus infectivity and neutralizing antibody assays

In a collaborative study sponsored by the European Commission for the Control of Foot and Mouth Disease, workers in 19 laboratories participated in the early phases (1, 2 and 4). All three phases were devoted to assessments of virus infectivity and the neutralizing activity of sera. Virus preparations and antisera distributed from one laboratory were tested either by 'in house' or suggested methods. Analyses of the results clearly showed that whilst 'within' laboratory variation in the results of replicate tests was low, there were significant differences in the results from various laboratories. By attempting to standardize test conditions with distributed materials, e.g. media and cells, etc., the 'between' laboratory variation was reduced.     

Standardizing global gene expression analysis between laboratories and across platforms

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

An international collaborative study of an anti-infectious bursal disease virus reference serum

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.     

Taste discrimination vs hedonic response to sucrose in coffee beverage. An interlaboratory study

Degree of liking, using a 17-point hedonic scale, and discriminationtaste thresholds, using a paired-comparison technique, weredetermined in a coffee beverage containing 0, 2.5, 5.0, 7.5,and 10.0% sucrose at laboratories in Brazil, Japan, Poland,Sweden and USA. Hedonic responses from the 122 subjects weresubdivided into four distinct sub-groups, according to differentpatterns as a function of sucrose concentration. Different frequenciesof these hedonic patterns resulted from the five laboratories.With few exceptions, repeated hedonic testing at the terminationof the experiment matched those from the beginning, indicatingstability of response during the lengthy study. No differenceswere observed in hedonic responses (nor in discrimination ability)between the male and female subjects at each laboratory. Discrimination thresholds at the five standard sucrose concentrations,and corresponding Weber ratios, were reported for the pooleddata within each laboratory. In general, the Weber ratios werehigher at the lower concentrations, indicating dependence ofdiscrimination upon the standard concentration. Notable differencesin discrimination ability were evident among the five laboratories,but were unrelated to degree of liking for sweetness in thecoffee. Subjects with low or with high degree of liking forall coffee samples, as well as those with increasing or decreasinghedonic responses as a function of sucrose concentration, discriminatedequally well among the concentration levels. The data from alllaboratories showed that ability to discriminate among sucroselevels and degree of liking for sucrose levels in coffee areindependent behavioral responses. ¹Present address: Department of Nutrition, Cornell University,Ithaca, New York, USA     

Comparison of Methods for Coccidioidomycosis Complement Fixation

A Laboratory Branch Task Force of the National Communicable Disease Center has proposed a standardized complement fixation procedure (LBCF) and an adaptation of this to microtitration techniques (MT) as uniform methods for performing complement fixation (CF) tests. A common procedure should make CF results from one laboratory more comparable to another. In addition, it would be preferable if the common procedure reproduced the titer levels of a testing procedure which is to be replaced, particularly when valid clinical interpretations have been derived from the latter. Replicated sets of sera were tested by the LBCF, MT, and the standard Smith CF procedure for coccidioidomycosis. Results with all three procedures were highly reproducible within an acceptable one-tube variation of a twofold dilution series, but the frequency of one-tube variations was greater with the MT method than with the other two. There was no statistical difference in the titers obtained with the Smith and LBCF procedures, but there was a significant difference when the MT results were compared to those with the Smith method. The LBCF method should be acceptable as a standardized and uniform CF procedure for coccidioidomycosis, subject to comparative testing between different laboratories.     

Sources of variation in the mutagenic potency of complex chemical mixtures based on the Salmonella/microsome assay.

Twenty laboratories worldwide participated in a collaborative trial sponsored by the International Programme on Chemical Safety on the mutagenicity of complex mixtures as expressed in the Salmonella/microsome assay. The U.S. National Institute of Standards and Technology provided homogeneous reference samples of urban air and diesel particles and a coal tar solution to each participating laboratory, along with samples of benzo[a]pyrene and 1-nitropyrene which served as positive controls. Mutagenic potency was characterized by the slope of the initial linear component of the dose-response curve. Analysis of variance revealed significant interlaboratory variation in mutagenic potency, which accounted for 57-96% of the total variance on a logarithmic scale, depending on the sample, strain and activation conditions. Variation among replicate extractions of organic material (required for the air and diesel particles) and among replicate bioassays within the same laboratory was also appreciable. The average potencies for air and diesel particles in laboratories using Soxhlet extracts were not significantly different from those in laboratories using sonication, although there was larger interlaboratory variation for the Soxhlet method. Repeatability (which approximates the coefficient of variation within laboratories) ranged from 18 to 40% for air and diesel particles extracted using sonication, depending on the strain and activation conditions. Repeatability of Soxhlet-extracted air and diesel particles, however, ranged from about 37 to 89% including outliers and from about 11 to 31% excluding outliers. Repeatability of the coal tar sample and the 2 positive controls was in the range 18-34%. Reproducibility (which approximates the coefficient of variation between laboratories) was generally at least twice repeatability, and exceeded 100% for Soxhlet-extracted air and diesel particles, as well as 1-nitropyrene. Reanalysis of the data omitting observations of more than 1500 revertants/plate generally had little effect on these results. Elimination of outlying observations had limited impact, with the exception of Soxhlet-extracted air and diesel particles. In this case, reproducibility of bioassay results was notably improved, due largely to the omission of results for replicate extractions which varied more than 5-fold within one laboratory. Normalization of the log potency slopes for the mixtures by the corresponding slopes for benzo[a]pyrene tended to reduce this variation, although variation was increased after normalization by 1-nitropyrene. Adjustment for the percentage of organic matter extracted from the air and diesel particulate samples had little effect on variation for sonication-extracted particles, whereas variation was reduced for diesel particles and increased for air particles for Soxhlet.     

Evaluation of a trapping ELISA for the differentiation of foot-and-mouth disease virus strains using monoclonal antibodies.

A trapping enzyme-linked immunosorbent assay (ELISA) has been evaluated for the differentiation of foot-and-mouth disease virus (FMDV) strains using a panel of seven anti-serotype O monoclonal antibodies (MAbs). The variation of results within and between tests performed on the same day and on different days was examined using three strains of FMDV. Criteria for establishing antigenic differences between the strains as defined by the individual MAbs are proposed based on the variability measured, which can be used as standards by workers performing this test with other MAbs and FMDV strains.     

Fractionation of rat fibroblasts in a zonal rotor by means of a viscosity barrier.

Cell fractionation procedures involving differential sedimentation followed by resuspension of pellets and isopycnic centrifugation are very difficult to apply to the small amounts of material available from tissue culture cells. We have explored the possibility of successive differential and isopycnic sedimentation in a zonal rotor using a short viscosity barrier for the differential sedimentation. The marker enzymes used were cytochrome oxidase, acid phosphatase, catalase, and 5′-nucleotidase. The results of these procedures are compared to the results of one-step isopycnic separations in gradients of sucrose and Stractan. The Stractan gradient was much more effective than the sucrose gradient in separating the marker enzymes from the proteins of a postnuclear supernatant, but neither type of gradient could significantly purify the marker enzymes one from another. A two-step procedure using a viscosity barrier was effective in separating particles carrying catalase from the other marker enzymes assayed and from most of the protein. A three-step procedure resulted in similar purification of mitochondria. Modification of barrier composition and centrifugation times would probably result in further improvement of separations according to individual requirements for yield, purification, and freedom from specific contamination by other subcellular particles.     

Round robin investigation of methods for the recovery of poliovirus from drinking water

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavioral disinhibition by mescaline.

Rats were exposed to a CER procedure in which sucrose drinking was suppressed by a tone previously paired with shock. Suppression of drinking during the tone was reduced by mescaline (50 mg/kg) independently of whether training took place under mescaline or placebo. Additional data on the effect of mescaline on sucrose drinking indicated that the result could not be attributed to an increased drive to drink sucrose. It was proposed that mescaline releases behavior from inhibitory control. A number of studies from the literature were cited which supported this hypothesis.     

Physicochemical methods for predicting the biological potency of recombinant follicle stimulating hormone: an international collaborative study of isoelectric focusing and capillary zone electrophoresis.

Two methods for predicting the specific in vivo bioactivity of recombinant follicle stimulating hormone (FSH), based on quantitative measures of isoform distribution by isoelectric focusing (IEF)(1) and by capillary zone electrophoresis (CZE)(2) respectively, have been subjected to an international collaborative study by six laboratories from six countries. Both methods were used to estimate the predicted bioactivities of four preparations of follitropin beta, coded FSH A-D, differing widely in their isoform compositions and specific bioactivities. The mean predicted estimate of potency by IEF and CZE for each FSH preparation by each laboratory was within 80-125% of its potency estimated by bioassay, except for the mean estimates by CZE of that for FSH A by one laboratory and of that of FSH D by another. Each of the six laboratories using the IEF method, and each of the five laboratories using the CZE method were able to rank these FSHs according to their bioactivities, namely FSH B>FSH C>FSH A>FSH D. All laboratories were able to use both IEF and CZE to discriminate between FSH A and C, with bioactivities within 76-132% of one another. Four of six laboratories were able to use IEF, and two of five laboratories were able to use CZE, to discriminate between FSH B and C, with bioactivities within 89-112% of one another. This suggests that the accuracy and precision of both these methods should be sufficient to discriminate between FSHs which would meet or fail European Pharmacopoeia requirements for this type of hormone, since these stipulate that estimates of potency should fall between 80-125% of its stated potency. Using in most cases duplicate estimates in two independent assays, and excluding Laboratory 4, the pooled intra-laboratory geometric coefficient of variation (GCV) was about 4% for both IEF and CZE, and the inter-laboratory GCV was about 7% for IEF and about 10% for CZE. The use of one FSH preparation as a standard, with its specific activity as an assigned value, reduced the inter-laboratory variability of estimates for the remaining FSHs by both methods. This increased the accuracy of the predicted estimates of bioactivity for these remaining FSHs in terms of their approximation to the values for their bioactivities estimated by bioassay. These data therefore suggest that both these methods, and particularly IEF, are sufficiently accurate, precise and robust to be used for predicting the bioactivity of batches of follitropin beta, and especially if used with a standard preparation.     

A critique of the collaborative cytogenetics study to measure and minimize interlaboratory variation.

A statistical reanalysis was performed on the data fecently reported on a 6-laboratory, collaborative cytogenetic study to measure and minimize interlaboratory variation. Three of the laboratories had mean values significantly different from the others on most of the 6 indexes of chemically-induced aberration; one laboratory with values higher and two with values lower. Furthermore, relative variability of the values around the means was consistently lower in one of the 6 participating laborabories. The results of the reanalysis of this collaborative study demonstrates that significant interlaboratory differences exist and that these should be adjusted or diminished before rat cytogenetic analysis can be an effective test system for evaluation of a compound for mutagenic potential.     

麻疹减毒活疫苗国家参考品的制备及标定

为了制备麻疹减毒活疫苗国家参考品,选用国内麻疹疫苗生产株沪191制备麻疹疫苗参考品。生产过程中严格控制精密性、水分含量,对候选参考品进行鉴别试验、水分含量、病毒滴度及无菌检查等检验。检验合格后组织进行候选参考品病毒滴度协作标定,共有5个实验室参加了协作标定。协作标定完成后,对实验室内变异、实验室间变异及国际参考品在不同实验室间的变异进行了分析。此外还对疫苗进行了热稳定性和实时稳定性分析。结果显示经5个实验室协作标定后,麻疹减毒活疫苗国家参考品的滴度为4.96±0.26 lgCC ID50/m l,实验室内部变异在1.09%～4.64%之间,实验室间变异为2.62%。国际参考品在不同实验室间的变异为4.19%。稳定性考核数据表明制备的参考品具有较好的稳定性,符合作为麻疹减毒活疫苗国家参考品的要求,滴度为4.96±0.26 lgCC ID50/m l。