Prediction of protein structural classes by a new measure of information discrepancy

Since it was observed that the structural class of a protein is related to its amino acid composition, various methods based on amino acid composition have been proposed to predict protein structural classes. Though those methods are effective to some degree, their predictive quality is confined because amino acid composition cannot sufficiently include the information of protein sequences. In this paper, a measure of information discrepancy is applied to the prediction of protein structural classes; different from the previous methods, this new approach is based on the comparisons of subsequence distributions; therefore, the effect of residue order on protein structure is taken into account. The predictive results of the new approach on the same data set are better than those of the previous methods. As to a data set of 1401 sequences with no more than 30% redundancy, the overall correctness rates of resubstitution test and Jackknife test are 99.4 and 75.02%, respectively, and to other data sets the similar results are also obtained. All tests demonstrate that the residue order along protein sequences plays an important role on recognition of protein structural classes, especially for alpha/beta proteins and alpha+beta proteins. In addition, the tests also show that the new method is simple and efficient.     

A study on LH- and FSH-RIA kits by immunoradiometric assay

An evaluation of LH- and FSH-RIA BEAD kits based on IRMA was carried out. The results obtained with the methods characterized by the use of monoclonal antibodies, i.e., one is linked to solid phase, and another is isotopically labeled, were compared with those determined by the Daiichi LH- and FSH-kits. Intra- and inter-assay precision, recovery, linearity, and specificity of both methods were favorable without exceptions. The cross reactivity of the LH kit to 5,000 mIU/ml hCG revealed within the range of less than 3 mIU/ml. Significant correlations were observed between the results derived from conventional Daiichi LH- and FSH-kits. The results from the conventional kits exhibited 30 to 40% of those from the Daiichi kits, considered to be mainly due to the difference in standard calibrations used. Among the individuals within the normal menstrual cycle, the serum LH- and FSH-levels determined by the present kits gave a typical pattern with a peak in the preovulatory phase. On the other hand, the LH- and FSH-values of individuals in normal pregnancy revealed strikingly decreased in comparison with those of non-pregnant women.     

Normalisation of data from allergens proficiency tests

The problem of allergen analysis using ELISA kits from different commercial products giving significantly different results is widely acknowledged. The effect on proficiency testing results is that different assigned values have to be generated for the different kits used. Some experimental Food Analysis Performance Assessment Scheme (FAPAS) proficiency tests aimed to establish whether the use of a standardised calibrant could be used to normalise the complete data set without recourse to differentiation. Three recent FAPAS proficiency tests (2776 peanut, 2778 soya and 2781 gluten) sent out a second spiked sample, in addition to the usual spiked and unspiked samples. Further analysis of the data was undertaken after the completion of the tests. The ratio of the submitted results for the two spiked samples yielded complete data sets which could be tested for normality of the distribution. Where the raw data for each individual test sample was clearly non-normal and multi-modal, the ratio data yielded a much more normal and symmetrical distribution. The use of one of the test samples as a single-point calibrant has some limitations but the principle of applying a standardisation clearly works. The development of internationally recognised sets of certified reference calibration standards for use by allergens testing laboratories would greatly benefit the analysis.     

Dose‐Dependent Response of Personal Glucose Meters to Nicotinamide Coenzymes: Applications to Point‐of‐Care Diagnostics of Many Non‐Glucose Targets in a Single Step

We report a discovery that personal glucose meters (PGMs) can give a dose‐dependent response to nicotinamide coenzymes, such as the reduced form of nicotinamide adenine dinucleotide (NADH). We have developed methods that take advantage of this discovery to perform one‐step homogeneous assays of many non‐glucose targets that are difficult to recognize by DNAzymes, aptamers, or antibodies, and without the need for conjugation and multiple steps of sample dilution, separation, or fluid manipulation. The methods are based on the target‐induced consumption or production of NADH through cascade enzymatic reactions. Simultaneous monitoring of the glucose and L ‐lactate levels in human plasma from patients with diabetes is demonstrated and the results are comparable to those from current standard test methods. Since a large number of commercially available enzymatic assay kits utilize NADH in their detection, this discovery will allow the transformation of almost all of these clinical lab tests into POC tests that use a PGM.     

The use of centrally prepared reagents in an external quality experimental trial.

The authors provide the results of a short-term experimental trial in external quality assessment in 42 clinical laboratories conducted by the Mexican Ministry of Health. Assay kits for glucose, urea, and creatinine were prepared by the Ministry. The results may prove useful to organizers of external quality assessments in third-world countries who may opt for this strategy to improve performance. The laboratories performed the tests on reconstituted lyophilized control serum, also prepared by the Ministry. All three assays were performed manually using colorimetric methods. On the basis of their intralaboratory precision (coefficients of variation less than 8%, 8-12%, and greater than 12% for high, medium, and low precision, respectively), 12 laboratories demonstrated high precision for all three tests. Eight laboratories showed medium and low precision for different tests, while the other 22 fell in between. The results showed that the strategy of using centrally prepared reagents to improve interlaboratory agreement did not work well for urea and creatinine, but met expectations for glucose. The laboratories achieved an interlaboratory coefficient of variation of 10% for glucose in this first trial.     

Validation of two commercial lateral flow devices for the detection of peanut proteins in cookies: interlaboratory study

Results are reported for an interlaboratory validation study of 2 commercially available Iateral flow devices (dipstick tests) designed to detect peanut residues in food matrixes. The test samples used in this study were cookies containing peanuts at 7 different concentrations in the range of 0-30 mg peanuts/kg food matrix. The test samples with sufficient and proven homogeneity were prepared in our laboratory. The analyses of the samples (5 times per level by each laboratory) were performed by 18 laboratories worldwide which submitted a total of 1260 analytical results. One laboratory was found to be an outlier for one of the test kits. In general, both test kits performed well. However, some false-negative results were reported for all matrixes containing < 21 mg peanuts/kg cookie. It must be stressed that the test kits were challenged beyond their cut-off limits (> or = 5 mg/kg, depending on the food matrix). One test kit showed fewer false-negative results, but it led to some false-positive results for the blank materials. The sensitivity of the dipstick tests approaches that achieved with enzyme-linked immunosorbent assays.     

Comparison of AM1 and PM3 semiempirical to ab initio methods in the study of Diels-Alder reactions of butadiene and cyclopentadiene with cyanoethylenes

Assuming a concerted synchronous mechanism with one transition state of the Diels-Alder reactions, the structures of the transition states and the activation energies for the reactions of butadiene and cyclopentadiene with cyanoethylenes were calculated by AM1 and PM3 semiempirical methods. The structural parameters were compared with those obtained by high level Gaussian calculations, whereas the activation energies were compared both with the ab initio calculations and those obtained experimentally. The structural properties calculated with PM3 methods are in general in better agreement with the ab initio calculations. The low level ab initio calculations are in many cases worse than the semiempirical methods. All predicted activation energies with both semiempirical methods are up to 300% higher than the experimental values. The predicted reactivity is also opposite to the experimental data. Only the very high level Gaussian calculations are in good correlation with experimental results. The predicted selectivity of the reaction is also opposite to the experimental facts. Two explanations are offered for this discrepancy: AM1 and PM3 methods cannot handle the calculation of the concerted Diels-Alder transition states and are not recommended to be used for that purpose, or this Diels-Alder reaction is not concerted but is stepwise.     

Immunoreactivity and detection of wheat proteins by commercial ELISA kits

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.     

Proficiency tests to evaluate commercially available IVD kits for glucose and cholesterol measurements

The first proficiency testing round 630-IL-1002, was carried out with a Reference Material DMR-180a with reference values obtained by using gas chromatography isotope dilution mass spectrometry methods, in which glucose, cholesterol and creatinine were measured. The serum pool was obtained from blood donors and all the analytes were at the normal concentration in Mexican population. The laboratories participants used different field methods to measure the analytes. The Mexican compulsory standard NOM-064-SSA1-1993 “specifications for equipments in vitro diagnostic (IVD)” requests 5% precision and 5% maximum bias of the IVD equipments in the measurements of analytes like glucose and cholesterol. The results obtained by field laboratories in the proficiency testing round are compared to the reference value and uncertainty provided by the National Metrology Institute (CENAM). The quality of measurements is dependent not only on the laboratory competence but also on the methods used by those commercially available IVD kits. It is concluded that quality assessment of measurements in clinical laboratories should be critically evaluated by using stable and certified reference materials. Presented at MEFNM 2008, September 2008, Budapest, Hungary.     

Evaluation and comparison of high-sensitivity immunoradiometric assay kits for thyroid stimulating hormone

Fundamental and clinical characteristics of 3 kinds of high-sensitivity immunoradiometric assay (IRMA) kits for thyroid stimulating hormone (TSH). i.e., RIA BEADS II (kit A), TSH kit Daiichi II (kit B) and Ab tube TSH 'Eiken' (kit C) and one conventional radioimmunoassay (RIA) kit, i.e., TSH kit Daiichi (kit D), were studied. In the recovery test and the reproducibility test, there was no significant difference between the 4 kits. The sensitivities of kits A, B and C were much higher than that of kit D, and those IRMA kits were sensitive enough to distinguish hyperthyroidism from normal samples. For low concentrations of TSH (less than 5 microU/ml), the data from kits D, B, C and A tended to show higher values in that order. The correlation between the data measured by kits B and D, and the tendency of kit A toward lower values agreed well with other reports.     

Strategy of analytical test kits

The importance of test kits is increasing due to economical reasons. The different types of test kits allow qualitative, semiquantitative or quantitative results depending on the requirements. For applications where rapidity and mobility are absolutely necessary, they have advantages compared to laboratory methods. This is demonstrated by two typical examples. Besides the advantages, the limits of test kits are also mentioned. Test kits look very simple and are really easy to handle; nevertheless, not only the producers but also the users have to fulfil some basic demands.     

Strategy of analytical test kits

The importance of test kits is increasing due to economical reasons. The different types of test kits allow qualitative, semiquantitative or quantitative results depending on the requirements. For applications where rapidity and mobility are absolutely necessary, they have advantages compared to laboratory methods. This is demonstrated by two typical examples. Besides the advantages, the limits of test kits are also mentioned. Test kits look very simple and are really easy to handle; nevertheless, not only the producers but also the users have to fulfil some basic demands.     

The use of split-sample design for performance evaluation of screening kits

Laboratory medicine is an important discipline in health care with its remarkable effect on risk assessment, diagnosis of health, and disease state. This accounts describes a newborn screening approach involving retest, recall, and follow-up procedures. This real life trial emphasizes the need for split-sample design evaluation of newly opened test kits. Quantitative measurements of phenylalanine and neonatal thyroid stimulating hormone (nTSH) were performed in two laboratories. After validation of the calibration in the laboratory that was using the industrially prepared screening kits for the first time, the same real newborn blood spot samples were analyzed for phenylalanine and nTSH measurements in both laboratories and the results obtained were compared non-parametrically, and examined by Deming regression and difference plots. There was no problem with the phenylalanine results: similar results were obtained for the same blood spot cards in both laboratories (P=0.496; bias estimation 0.13). However, the nTSH values were found to be significantly higher in the laboratory that used the nTSH kit for the first time. Although the validation of the calibration of the nTSH kit was valid with the manufactures control materials, spilt-sample results showed that there was a significant difference between the two laboratories (P=0.005; bias estimation 28.6). This study implies that acceptable comparability of split-sample design analysis is needed for testing the analytical performance of industrially prepared tests kits, and this can only be achieved with certified reference materials.Presented at the 8th Conference on Quality in the Spotlight, 17–18 March 2003, Antwerp, Belgium     

The effect of some possible factors in c-peptide immunoreactivity in urine on the quality of the results

About 15% of CPR-U did not correspond between two kinds of kits in the routine assay, which were produced by different pharmaceutical firms, and also it is realised that the effect of the preservative, storage condition and buffer dilutions can not be overlooked in quality control programmes. Based on these problems, following results are concluded; (1) Urine preservative: NaN3 is better than toluene. (2) Urine CPR was stable for added preservative at 4 degrees C for two weeks. (3) Diluents may effect the CPR-U results, if not kept in good condition. The role of the makers in supporting improved quality in clinical laboratory tests will be expected.     

A COMPARISON OF IN VIVO AND IN VITRO TESTING OF SUNSCREENING FORMULAS

Abstract— Seven commercially available sunscreens were compared by three different methods. Absorbance spectra were measured for each product in isopropanol solution and also on hairless mouse epidermis. In vivo tests were performed on human volunteers using a Xe arc solar simulator. Sun Protection Factors (SPF) were calculated by each method for each product tested and the results compared. By all methods used, the combination of 7% octyl dimethyl para-aminobenzoic acid and 3% oxybenzone provided the most protection from U.V. light. While estimates of the effectiveness of all products were much too high when calculated by the isopropanol solution method, the hairless mouse epidermis technique seems to be an accurate tool for predicting product efficacy in vivo .     

In vivo measurement of nitrogen by NAA

Nitrogen in the human body is measured in vivo using prompt neutron capture gamma-rays. The quantity of nitrogen can then be used as measured of protein. Data are presented on three groups of subjects; volunteers of different ages, those with liver ailments, and those on peritoneal dialysis. The data show that the nitrogen measurements given information (in accord with clinical findings) which is not given by indirect methods of estimating lean body mass.     

Verification of the Conditions for Determination of Antioxidant Activity by ABTS and DPPH Assays—A Practical Approach

The ABTS and DPPH methods are among the most popular assays of antioxidant activity determination. Attempts to adapt them to different analytes and the search for the highest values of antioxidant activity has resulted in a large variety of assay conditions to be presented in the literature, including the way the measurement is made. This makes it difficult to relate the results to real oxidation systems, and often makes it impossible to compare them. Such a comparison is limited in advance by the use of stable radicals that do not exist in nature and that react differently from those generated in food or in vivo. Therefore, it is important to introduce measures aimed at standardizing the conditions of the activity assay, including reaction time and several reaction environments suitable for testing different groups of compounds. In this study, we used natural antioxidants of various structures: phenolic acids, flavonoids, peptides and corresponding amino acids, ascorbic acid and α-tocopherol, and also synthetic analogues of selected compounds. The curves of dependence of the measured absorbance on the concentration of antioxidants were described, the ranges of linearity were determined, and the value of the error made when reading in various ranges of dependencies was estimated. We also determined and compared the activity values using two popular methods (IC₅₀ and TEAC), taking into account different environments and reaction times. Based on the collected data, recommendations were formulated regarding the reaction conditions adapted to the studies of individual groups of antioxidants, and unified reaction times were proposed. Taking into account the state before reaching the equilibrium of antioxidants reacting in a complex manner, this approach may introduce a simplified reference to the competing reaction that occurs in reality.     

Towards standardization of external quality assessment schemes by using bias values based on biological variation

The precision and trueness of current instruments and methods in clinical laboratories is much better than in the past. However, the z-score and other comparison variables that are currently used in external quality assessment programs are based on relative data. Thus, they may change from program to program and also change over time within the same program and consequently may not be useful or cost-effective. We therefore devised a test-specific decision limit for accepting or rejecting test results based on a combination of the data of within- and between-subject biological variations. We then applied these limits to a group of tests performed in our laboratory and compared our results with those of external quality assessment programs. In addition, we combined external and internal quality control data on the same graph and prepared a two-dimensional graph for different levels of control sera. Inspection of all results of control sera on this new graph was more useful for decision making. We concluded that the z-score is not reliable for comparisons of test results in external quality assessment. As a substitute for this currently accepted practice, we assert that control limits based on biological variation are more reliable, and can be useful in the evaluation of both external and internal quality assessments and their combination on this new graph.     

Accuracy in naturally occurring anabolic steroid assays in cattle and first approach to quality control in Italy

This study assessed the accuracy of 16 commercial and three self-produced kits and drew the basis for using an external quality control (EQC) system. The commercial kits were mainly developed for blood sex steroid determination in humans but also have been used in cattle. Parallelism, recovery and precision tests were performed for progesterone (P4), testosterone (T) and oestradiol (E2) assays. Moreover, anonymous QC samples were sent to be analysed to some Italian laboratories. All kits showed a fair degree of parallelism (P < 0.01), even though 2/7 kits for T and 1/6 kits for E2 determination showed a regression coefficient (r2) lower than 0.98. For P4, an acceptable range of accuracy was achieved in the recovery test only by 1/6 kits; two kits showed fair or great overestimation and two kits considerable underestimation. For T, an acceptable range of accuracy was achieved only by 1/7 kits. For E2, 4/6 kits presented a variable degree of underestimation and two kits showed great overestimation. In the intra-assay precision test quite good repeatability was achieved only using samples with high hormone concentrations. While assaying samples with low concentrations we found a number of RSD > 10%. Moreover, the laboratories participating in the EQC produced statistically different (P < 0.05) results, particularly for high and medium concentrations. In conclusion, the use of commercial kits for screening naturally occurring sex steroid concentrations in cattle blood, in the case of suspected illegal treatments, requires preventive validation procedures and the development of an opportune EQC system.     

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency,fragment size bias and quantification

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp? plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. Figure

Comparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA