A new luminescence immunoassay for thyrotropin using coated tubes: Evaluation and comparison with immunoradiometric assay

A recently available chemiluminescence immunoassay (LIA-MAT) for TSH, set up by Byk-Sangtec Diagnostica, has been evaluated and compared with IRMA methods. The system LIA-MAT uses two monoclonal antibodies: one coated on tubes and the other labelled with isoluminol. To compute the within-assay precision profile, we estimated the response error relationship from all the duplicates (420) of eleven experiments. The CV of the response was 4–5% from the maximal response until 10,000 RLU (corresponding to about 1 μlU/ml); in the lower response range the CV worsened up to 8%. The sensitivity, derived from the precision profile, was 0.052 μlU/ml similar to that found in IRMA-MAT (0.044 μlU/ml); the working range extended from 0.33 to 100 μlU/ml. Results from LIA-MAT in the concentration range 1–30 μlU/ml were compared with the consensus mean produced by users of IRMA methods (IRMA-MAT, IRMA-Behring, Maiaclone Serono) participating in an inter-laboratory survey; a good correlation with IRMA techniques was found. The distribution of TSH determinations produced by LIA-MAT on 62 low concentration sample (<0.3 μlU/ml) from patients unresponsive to TRH test, was found similar to that observed for the kit IRMA Boots-Celltech assumed as reference.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Prenylated flavonoids and total extracts from Morus nigra L. root bark inhibit in vitro growth of plant pathogenic fungi

Total extracts and kuwanon G from Morus nigra root bark showed antifungal activity against several phytopathogenic fungi, with minimal inhibitory concentration (MIC₅₀) ranging from 32 to 128 μg/ml and from 16 to 64 μg/ml, respectively. Acetonic extracts inhibited 60% B. cinerea biofilm formation at concentration of 128 μg/ml.     

Quantification of 1,5-anhydro-d-glucitol in urine by automated borate complex anion-exchange chromatography with an immobilized enzyme reactor

HPLC using a borate form of a strongly anion-exchange resin column and an immobilized enzyme reactor for colorimetric detection was used to quantify urinary 1,5-anhydro-d-glucitol. Urine samples were introduced into the system every 7 min without any pretreatment, and after separation of interfering substances in the column, 1,5-anhydro-d-glucitol was successively detected. Quantitative determination of urinary 1,5-anhydro-d-glucitol was possible within the 1.2–300 μmol/l range. The coefficient of variance was less than 3% and the correlation between results obtained with our system (y) and those obtained by gas chromatography–mass spectrometry (x) was y=0.983x−1.287 μmol/l (n=42, r=0.998).     

A New Functional Equation Analogous to CAUCHT-PEXIDER Functional Equation and its Application

The CAUCHY-PEXIDER functional equation H (x±y)=F(x) G(y) is generalized to the form H ((x^c±y^c)^1/c) = F(x) G(y), c≠0, assuming the function H(x) possesses a measurable majorant on a set of positive measure. The result is used to obtain a characterization of WEIBULL distribution. This functional equation is generalized to functions of vector variables.     

Steroids and triterpenoids from Eastern Nigeria mistletoe, Loranthus micranthus Linn. (Loranthaceae) parasitic on Kola acuminata with immunomodulatory potentials

In search of immunomodulatory constituents from the Eastern Nigeria mistletoe, Loranthus micranthus Linn, two new stigmastane steroids: stigmast-7,20 (21)-diene-3β-hydroxy-6-one (1) and 3β-hydroxy-stigmast-23-ene (2); three (two new and one known) lupeol-based triterpenoid esters: 7β,15α-dihydroxyl-lup-20(29)-ene-3β-palmitate (3), 7β,15α-dihydroxyl-lup-20(29)-ene-3β-stearate (4) and 7β,15α-dihydroxyl-lup-20(29)-ene-3β-eicosanoate (5) were isolated and characterized following bioactivity-guided fractionation. The new compounds, 1, 2, 4 and 5 at concentrations of 10, 25 and 100 μg/ml were subjected to cell proliferation and early activation marker (CD69) expression studies in C57Bl/6 mice splenocytes using flow cytometry techniques against Lipopolysaccharide (LPS; 10 μg/ml) and Concanavalin A (ConA; 2 μg/ml) standards. The stigmastane steroids (1 and 2) at the highest concentration of 100 μg/ml showed statistically significantly (p < 0.05) stimulatory activity on the C57B1/6 splenocytes compared to the controls with values of 46 ± 0.76% and 43 ± 0.46% compared to 7.69 ± 0.41% recorded for the negative control. The novel lupeol esters, 4 and 5 at same concentration of 100 μg/ml exhibited lower stimulations of 30 ± 0.41% and 29 ± 0.17% respectively compared to the controls above. The CD69 expression assay at the above doses showed that all the compounds have minimal stimulation. The present study supports the observed immunomodulatory property of the Eastern Nigeria mistletoe and thus confirms the efficacy of this plant in mitigating against wide array of disease conditions orchestrated by immunodeficiency.     

An enzyme-linked immunoassay for the levansucrase ofZymomonas mobilis using specific antibodies produced against the cloned enzyme

Summary Levansucrase gene (levU) ofZ. mobilis had been cloned and overexpressed inEscherichia coli (Song, K.-B. and Rhee, S.-K. (1994)Biotechnol. Lett. 16, 1305–1310). The levansucrase was purified and specific antibodies against it were raised in rabbits. Specificity of the antibodies was demonstrated by Western analysis. By using the antibodies, an efficient, enzyme-linked immunosorbent assay (ELISA) amenable to immunochemical analysis of the levansucrase has been developed. The working range of levansucrase concentration in the developed ELISA was between 75 and 300 ng/ml. The assay was very sensitive to detect the levansucrase as little as 37 ng/ml.     

Schistosomicidal Effects of the Essential Oils of Citrus limonia and Citrus reticulata Against Schistosoma mansoni

We report the in vitro schistosomicidal effects of the essential oil obtained from Citrus limonia leaves (CL ‐EO ) and C. reticulata fruit peels (CR ‐EO ), cultivated in Brazil, against Schistosoma mansoni worms. Limonene (29.9%), β ‐pinene (12.0%), sabinene (9.0%), citronellal (9.0%), and citronellol (5.8%) are the major constituents of CL ‐EO ; limonene (26.5%), γ ‐terpinene (17.2%), linalool (11.1%), octanal (8.0%), myrcene (6.2%), and capraldehyde (3.9%) predominate in CR ‐EO . CL ‐EO displayed moderate lethal concentration 50% (LC ₅₀) of 81.7 and 38.9 μg/ml against male and female worms at 24 and 72 h, respectively. At concentrations of 25 and 100 μg/ml, CL ‐EO separated between 50 and 75% of the coupled worm pairs during the evaluated period. CR ‐EO presented moderate LC ₅₀ of 81.7 μg/ml against male and female worms at 24 and 72 h. However, this oil separated coupled worm pairs more effectively than CL ‐EO and displayed lower cytotoxicity to GM 07492‐A cells (IC ₅₀ = 987.7 ± 88.9 μg/ml) as compared to CL ‐EO (IC ₅₀ = 187.8 ± 2.9 μg/ml). The enantiomers (+)‐(R )‐limonene and (?)‐(S )‐limonene did not affect S. mansoni adult worm pairs significantly. Taken together, these data indicate that CL ‐EO and CR ‐EO exhibit moderate in vitro schistosomicidal activity against adult S. mansoni worms.     

The effect of copper on length increase in Ascophyllum nodosum (L.) Le Jolis

The increase in length of Ascophyllum nodosum (L.) Le Jolis was measured in copper concentrations varying from 14–5000 μg/1 sea water. The growth was unaffected by concentrations of 33 μg/1 or less added for a period of 11 days, but at higher concentrations growth was gradually reduced. When the number of days (x) of exposure to a concentration (y) (y > 33 μg/1) is compared with z (the corresponding percentage reduction of growth rate), the data fit the equation z = 0.084 xy up to at least z = 60%.     

Composition and Activity against Oral Pathogens of the Essential Oil of Melampodium divaricatum (Rich.) DC.

The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich .) DC. (Asteraceae) was characterized by GC‐FID and GC/MS analyses. (E)‐Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 μg/ml were obtained against Streptococcus sobrinus (90 μg/ml), Lactobacillus casei (30 μg/ml), S. mutans (20 μg/ml), and S. mitis (18 μg/ml). In contrast, the MIC values of the major oil compound (E)‐caryophyllene were higher than 400 μg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.     

Bispecific-Ab-based immunoassay of thyroid-stimulating hormone

 A bispecific F(ab′)₂ fragment recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (AP) was prepared by disulfide bond exchange between F(ab′)₂ fragments of IgG1 mAb against TSH and AP. AP was polymerized by glutaraldehyde, and a sandwich enzyme-linked immunosorbent assay for TSH was developed by using the AP polymers and the bispecific F(ab′)₂ fragment. In this assay, the preparation of covalently linked AP-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced. The sensitivity for TSH increased in proportion to the degree of AP polymerization, and the lower detection limit obtained with the AP trimer was 0.5 μU/ml. The use of the bispecific F(ab′)₂ fragment allows us to use monomers and polymers of AP and thereby regulates the sensitivity of the assay. Accepted: 14 October 1997     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Use of Transformed Auxiliary Variable in Estimating the Finite Population Mean

For estimating the finite population mean Y- of the study character y, an estimator using a transformed auxiliary variable has been defined. The bias and mean-squared error (MSE) of the proposed estimator have been obtained. The regions of preference have been obtained under which it is better than usual unbiased estimator y-, the ratio estimator y-_R = y-X-/x-, Sisodia and Dwivedi (1981) estimator y-_s = y-(X- + C_x)/(x- + C_x) and Singh and Kakran (1993) estimator y-_k = y[X- + β₂(x)]/[x- + β₂(x)]. An empirical study has been carried out to demonstrate the superiority of the suggested estimator over the others.     

Determination of concentrations of flecainide in human serum by high-performance liquid chromatography on a fluorocarbon-bonded silica gel column

An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C₁₈ column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 μl; column temperature, 40°C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength=300 nm, emission wavelength=370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide–acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10–2000 ng/ml). The regression equation was y=0.08+0.0078x (r=0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94–100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.     

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.     

Determination of amikacin in human plasma by high-performance capillary electrophoresis with fluorescence detection

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length)×50 μm I.D.] at the anode side by dynamic compression injection (50 hPa for 6 s). After electrophoresis with 40 mM SDS-20 mM phosphate-borate buffer (pH 7) in the micellar electrokinetic chromatography (MEKC) mode at 30 kV, the derivative had a retention time of 16.7 min and was detected by fluorescence intensity at 482 nm (with irradiation at 414 nm). The precision (n = 5) of the method is 4.08 and 1.59% (C.V.) at the 50 and 100 μg AMK/ml plasma levels, respectively. Linearity (r = 0.998) was established over the concentration range 5–100 mg of AMK/ml plasma and the detection limit (at a signal-to-noise ratio of 3) is 0.5 μg AMK/ml plasma. This assay method could potentially have wider application in the determination of other aminocyclitol antibiotics, such as arbekacin, dibekacin, kanamycin, in human plasma as well as of AMK.     

Determination of mivacurium in plasma by high-performance liquid chromatography

An assay has been developed and validated for the routine monitoring of mivacurium in plasma. It consists of liquid-liquid extraction with dichloromethane and high-performance liquid chromatography with fluorometric detection (excitation and emission wavelengths 220 nm and 320 nm, respectively). A Spherisorb C₁ 5 μm column and a mobile phase containing acetonitrile, KH₂PO₄ and methanol are used. At a flow-rate of 1 ml/min, a concentration gradient is applied. The detection limit is approximately 1 ng/ml in plasma. For the separation of stereoisomeres, the Spherisorb SCX 10 μm column and acetonitrile-Na₂SO₄ as a mobile phase can be used. The assay shows good linearity over the range 1–1000 ng/ml. The accuracy and precision allows the utilisation in clinical pharmacokinetic studies.     

Optimal Fixing of the Bandwidth-Parameter for the Empirical Regression1,2

The estimator ?₀(x) of the regression r(x) = E (Y | × = x) from measured points (x_i, y_i), i = 1(1) n, of a continuous two-dimensional random variable (X, Y) with unknown continuous density function f(x, y) and with moments up to the second order can be made with the help of a density estimation f?₀(x, y) (see e.g. SCHMERLING and PEIL, 1980). Here f?₀(x, y) still contains free parameters (so-called band-width-parameters), the values of which have to be optimally fixed in the concrete case. This fixing can be done by using a modification of the maximum-likelihood principle including jackknife techniques. The parameter values can be also found from the estimators for r(x). Here the cross-validation principle can be applied. Some numerical aspects of these possibilities for optimally fixing the bandwidth-parameter are discussed by means of examples. If ?₀(x) is used as a smoothing operator for time series the optimal choice of the parameter values is dependent on the purpose of application of the smoothed time series. The fixing will then be done by considering the so-called filter-characteristic of ?C₀(x).     

Chemical Composition and Biological Activity of Centaurea baseri: New Species from Turkey

The genus Centaurea L. is one of the largest and important genera of Asteraceae family. Centaurea species have been widely used as herbal remedies in folk medicine for their antidandruff, antidiarrheic, antirheumatic, anti‐inflammatory, choleretic, diuretic, digestive, stomachic, astringent, antipyretic, cytotoxic, and antibacterial properties. Centaurea baseri Kose & Alan is a recently described local endemic species in Turkey and this is the first study on the chemical composition and bioactivity of its hydrodistilled essential oil and the crude extract. According to chromatospectral analysis, hexadecanoic acid (42.3%), nonacosane (8.2%), and heptacosane (8.0%) were the main compounds of the essential oil, while 16 compounds were determined in the MeOH extract using LC/MS. Furthermore, antimicrobial, antioxidant, and cytotoxic effects of the essential oil and the extract were evaluated in comparison with the standard agents. The extract showed strong antifungal effect against Candida utilis at the concentration of 60 μg/ml (MIC) where the EO showed growth inhibition at the concentration of 47.00 μg/ml (MIC) against pathogen Bacillus cereus. Both the essential oil and the extract did not show any selective antioxidant properties. The extract showed remarkably selective cytotoxic properties against MCF‐7, PANC‐1, A549, and C6 glioma cells.