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Biomedical subjects

G Giraudi

Publications and source records attributed to G Giraudi.

At least 19 recordsLinked to original sources
PubMed2006

Molecularly imprinted solid-phase extraction method for the high-performance liquid chromatographic analysis of fungicide pyrimethanil in wine.

A method for molecularly imprinted solid-phase extraction (MISPE) of the fungicide pyrimethanil from wine samples has been investigated. The molecular imprinted polymer was obtained by iniferter-mediated grafting on porous chloromethylated polystyrene beads, using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The imprinted beads were evaluated for use as a solid-phase extraction sorbent, in order to develop the extraction protocol in aqueous standards and red wine samples. The optimised extraction protocol resulted in a reliable MISPE method suitable for HPLC analysis (stationary phase: Cromolith Performance C18 column, 100 mm x 4.6 mm; mobile phase: acetonitrile-water (3:2, v/v), flow-rate: 1.00 ml/min; detection 270 nm). It was selective for pyrimethanil and the related pyrimidinic fungicides cyprodinil and mepanipyrim, while the non-pyrimidinic fungicides benalaxyl, chlozolinate, furalaxyl, iprodione, metalaxyl, nuarimol, procymidone and vinclozolin were not extracted. Recoveries performed on a wine matrix spiked with pyrimethanil at three different concentration levels were reproducible and were in good agreement with the recoveries performed on buffer, coming out between 80 and 90% (85+/-7.0% at 0.50 microg/ml, 79+/-1.6% at 2.0 microg/ml and 87+/-5.6% at 20 microg/ml). Preconcentration and quantitative extraction of pyrimethanil from wine samples was shown to be feasible down to 0.1 microg/ml.

Chromatography, High Pressure Liquid↗
PubMed2006

Antibody-like peptides as a novel purification tool for drugs design.

New pharmaceutical approaches, such as biotechnology industry, genomic and proteomic studies, require the development of new analytical and preparative tools that should allow the resolution and the characterisation of complex sets of molecule mixtures in a high-throughput mode with the isolation of a single substance from complex matrices in a high degree of purity, low costs and wide availability. In this review we discuss the design of a tailor-made peptide by focusing our attention on the bioinformatic techniques and combinatorial approaches. Then synthesis, purification and characterisation of peptides with recognition properties are discussed by considering different approaches and their fitness to drug design. Moreover, the development of affinity devices and the discussion of their characteristics to optimise the purification phase are reported. Applications of peptide ligands that bind pharmaceutical compounds (i.e. therapeutic proteins, monoclonal antibodies, hormones) are described and analytical tools mentioned and evaluated. Future perspectives, in particular alternative applications of peptide ligands and their substitution with peptidomimetic compounds, are described.

Animals↗
PubMed2001

A molecular imprinted polymer with recognition properties towards the carcinogenic mycotoxin ochratoxin A.

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L)-phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60 degrees C. The monolith obtained was crushed, sieved to 30-90 microm and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase. From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.

Carcinogens↗
PubMed2000

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid.

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed2000

Application of an ELISA to the determination of benalaxyl in red wines.

The applicability of an ELISA for detection and quantification of benalaxyl in red wine samples is described. The study of the influence of this matrix on the reliability of the assay indicates that red wine samples require a rapid and simple cleanup step before ELISA assay. Recovery and precision of the method were evaluated by spiking red wine samples with benalaxyl in the 0.5-24 ng/mL range. Benalaxyl can be determined with good accuracy and precision up to 0. 5 ng/mL in starting red wine samples (detection limit of 0.13 ng/mL). No false negative or positive results were obtained. Authentic red wine samples were analyzed by ELISA and by RP-HPLC. The amounts of benalaxyl found by ELISA were in good agreement with RP-HPLC analysis.

Alanine↗
PubMed1999

A general method to perform a noncompetitive immunoassay for small molecules.

A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.

Animals↗
PubMed1996

Strategy for fractionating high-affinity antibodies to steroid hormones by affinity chromatography.

A general strategy for fractionating high-affinity antibodies to steroid hormones has been developed and applied to the fractionation of an antiserum to testosterone 3-(O-carboxymethyl)oxime-bovine serum albumin. If the antibodies interacting with a stationary phase containing a low concentration of immobilized steroid are considered as monovalent binders, a simple equation can be applied to show that the affinity of the antibody-stationary phase interaction must be higher than about 2 x 10(6) l mol-1 in order to avoid the loss of antibodies during the loading and washing of the column. Conversely, to elute the retained antibodies, the affinity must be decreased to a value lower than about 2 x 10(5) l mol-1 and the dissociation rate constants of the antibody-steroid complexes must be >> 1 s-1. In order to prepare an affinity column that satisfies these conditions, the ligand to be immobilized was selected on the basis of the cross-reactions of the antiserum with several testosterone derivatives. Moreover, the dissociation rate constants of several antibodies of known affinity were measured, together with the effect of acidic buffers and various organic solvents on the antiserum-testosterone interaction. Then, an affinity column, prepared by coupling testosterone 17 beta-acetate to AH-Sepharose 4B, was used to load the antiserum without loss of antibodies during the washing step. The retained antibodies were successfully eluted by a mixture of 30% dioxane in phosphate-citrate buffer (pH 3.4). The affinity of the eluted antibodies was in the range 7 x 10(9)-2 x 10(11) l mol-1 and was linearly related to the retention volume. These results confirm that high-affinity antibodies can be fractionated to steroid hormones by a proper choice of the ligand on the stationary phase and the eluent composition.

Antibodies↗
PubMed1995

Fractionation of an antiserum to progesterone by affinity chromatography: effect of pH, solvents and biospecific adsorbents.

Several progesterone-AH Sepharose 4B matrices were prepared as biospecific adsorbents suitable for affinity chromatography to fractionate antibodies of different affinity and specificity from a polyclonal antiserum to progesterone-11 alpha-hemisuccinate-BSA. From an affinity column of progesterone-11 alpha-hemisuccinate-AH Sepharose 4B no antibodies can be eluted, even with glycine buffer (pH 2.6) and 30% of 2-methoxyethanol. The use of biospecific adsorbents, prepared by coupling with AH Sepharose 4B progesterone derivatives [5-pregnene-3,20-dione di(ethyleneacetal)-11 alpha-ol-11 alpha-hemisuccinate; 4-pregnene-11,20 beta-diol-3-one-11 alpha-hemisuccinate 20 beta-benzoate; progesterone-3-carboxymethyloxime] having a low cross-reactivity with the antiserum, makes the elution of various antibody fractions of variable affinity and specificity possible. 2-Methoxyethanol or N,N-dimethylformamide gradients, in acetate or TRIS buffer, were equally efficient for fractionating the antiprogesterone serum, while a decreasing pH gradient was less effective and eluted antibody fractions that were further separated into various binding components by a solvent gradient. Antibodies eluted from the affinity columns by an eluent containing a high solvent concentration have affinities higher than antibodies eluted at lower solvent concentration.

Absorption↗
PubMed1994

Immunochemical methods for environmental monitoring.

Immunochemical methods for environmental analysis must be taken into consideration more for their ability to expand the potential of analytical measures rather than for substituting current methodologies. Moreover, the full potential of these methods has yet to be realized. Indeed, the terms and concepts of immunology are new to most analytical chemists, even if environmental science has always been an interdisciplinary field. On the other hand, the clinical development of immunoassays means that much experience has been gained in the analysis of blood, urine, and tissue samples. The immunochemical analysis of samples from soils, ground water, waste chemicals, poses new challenges in sample preparation that have yet to be extensively studied, and in the future there may be immunoassays better suited for the particular problems associated with environmental monitoring.

Environmental Monitoring↗
PubMed1993

Solvent effect on testosterone-antitestosterone interaction.

The inhibition of the binding between testosterone and antitestosterone antiserum caused by organic solvents was studied at pH 7.4, 298 K. Inhibition curves were obtained at variable ranges of molar fractions for the following solvents: methanol (range 0-0.4), ethanol (0-0.317), 1-propanol (0-0.082), 2-propan-ol (0-0.260), t-butanol (0-0.223), ethylenglycol (0-0.189), 2-methoxyethanol (0.036), 2-butoxyethanol (0-0.063), 1,4-dioxan (0-0.124), tetrahydrofuran (0-0.238) and acetonitrile (0-0.392). Steroid-antibody binding decreases with increasing molar fraction of solvent in the reaction mixture for all but tetrahydrofuran and acetonitrile, which enhance binding at low molar fraction then cause a sharp inhibition. Molar fraction of solvent that causes a 50% binding inhibition is uncorrelated to some solvent properties (i.e. dielectric constant, polarity index, dipole moment) but is inversely correlated to the molecular mass of the solvent. The correlation becomes better by taking into account the length of the solvent molecule, or the Randic molecular connectivity index, suggesting that binding inhibition could be related to the length of the solvent molecules that displace water around the steroid molecule. However, the increase of binding observed at low molar fraction with tetrahydrofuran and acetonitrile, together with very different shapes of inhibition curves suggest that a molecular mechanism based on the differential solvation of the steroid by solvent and water molecules must be taken into account to explain adequately the solvent effect on testosterone-antitestosterone interaction.

Antibodies↗
PubMed1992

Transplacental passage of autoantibodies to triiodothyronine.

Autoantibodies to T3 (T3Ab) were detected by immunoprecipitation in a 18-year-old female patient affected by Graves' disease. The presence of these antibodies was constantly confirmed during a 7-year follow-up period, independently of therapy and functional thyroid status. Antithyroid microsomal antibodies (McAb) and TSH binding-inhibiting antibodies (TBII) were also present. The title of AbT3, McAb and TBII fluctuated, at times, independently of one another. The patient became pregnant; during pregnancy T3Ab concentration fell and went up again 4 months after delivery. A normal baby was born. T3Ab were present in the cord blood and declined during the following 2-8 months. The T3Ab of both mother and child belonged to the IgG class. The concentrations and the binding affinities of T3Ab in mother and child were identical. The presence of T3Ab in the child did not affect his thyroid function.

Adolescent↗
PubMed1990

The "soluble sandwich" approach for immunoassays: methodological and instrumental implications.

The simultaneous presence of homogeneous and heterogeneous reactions at different binding sites of a multiepitope antigen makes the description of the kinetic parameters of the so called "one step" solid phase immunometric assays complex. The authors extended the "one step" approach to the concept of the "soluble sandwich" methodology which differs from the former by the delayed solid phase capture of the biotinylated immunocomplex to a streptavidin coated solid support. Using prolactin monoclonal IEMAs as a model, the equilibria involved in the reactions have been studied on a thermodynamic basis through a description of the kinetics of the interactions between biotinylated Mabs and solid phase streptavidin both in presence and/or in absence of the antigen and HRP-conjugated antibody. A comparative evaluation of models in which the biotinylated antibody was previously insolubilized on the streptavidin solid phase has been performed as well. The experimental work was carried out by using 125I labelled McBiot and Prolactin to trace individual interactions and peroxidase/H2O2/TMB systems to develop the enzymatic analytical signals. A new instrument/data reducing system was also optimized to expand the OD reading range provided by conventional, single wavelength colorimeters. The greater flexibility theoretically expected for the "soluble sandwich" approach and the possibility to extend the analyte working range without detrimental effects on the readability of low doses responses have been experimentally confirmed.

Antibodies, Monoclonal↗
PubMed1988

Adrenocorticotropic hormone and catecholamines in maternal, umbilical and neonatal plasma in relation to vaginal delivery.

The aim of this study was to evaluate the effect of vaginal delivery on both ACTH and catecholamines (DA, NE, E) secretion in the mother, the fetus (umbilical artery) and the newborn. Blood samples were obtained from 19 normal pregnant women and the corresponding umbilical cords, and from the newborns. Seventeen normal nonpregnant women, matched for age and parity, were also included in the study as "nonpregnant controls". The results demonstrate that in the mother, plasma catecholamines (CA) concentrations during labor and delivery are elevated above the values reported for normal nonpregnant women and there is a predominant E response. The concentrations of CA in umbilical arteries are very high compared to those in the corresponding mother and they fall rapidly after birth. Unlike that in the mother, the predominant CA response to parturition in the fetus and newborn infant is NE. The extraction rate of DA, NE and E from placenta is approximately 60%. The peripheral plasma levels of ACTH in pregnant women during labor are twice and 10 times as high as those observed in the corresponding umbilical arteries and in nonpregnant women respectively. At delivery they increase further. No significant differences are found between the values measured in the arterial cord blood and those in the venous cord blood and in the newborns. A way of explaining the prevalence of E and the higher ACTH/E ratio found in the mother in comparison with the fetus could be that in the mother the stress response to parturition is regulated mainly by the pituitary-adrenal axis, whereas in the fetus there is a prevalent stimulation of the sympathetic nervous system.

Adrenocorticotropic Hormone↗
PubMed1987

Role of serum carrier proteins in the peripheral metabolism and tissue distribution of thyroid hormones in familial dysalbuminemic hyperthyroxinemia and congenital elevation of thyroxine-binding globulin.

To investigate the role of thyroxine-binding globulin (TBG) and albumin in the availability of thyroid hormones to peripheral tissues, comprehensive kinetic studies of thyroxine (T4) and triiodothyronine (T3) were carried out in eight subjects with familial dysalbuminemic hyperthyroxinemia (FDH), in four subjects with inherited TBG excess, and in 15 normals. In high-TBG subjects, the reduction of T4 and T3 plasma clearance rates (by 51% and 54%, respectively) was associated with normal daily productions; T4 and T3 distribution volumes were significantly reduced. In FDH subjects T4 clearance was less reduced (by 31%) than in high TBG; consequently T4 production rate was significantly increased (by 42%); T4 and T3 distribution volumes and T3 clearance rate were unchanged. Increased T3 peripheral production in FDH (by 24%) indicates that T4 bound to abnormal albumin is more available to tissues than T4 carried by TBG, thus suggesting an important role of albumin in T4 availability to the periphery.

Adult↗
PubMed1986

Development of thyroid function between VI-IX month of fetal life in humans.

Well-preserved thyroid glands from 28 fetuses 22-34 weeks of gestational age and from 4 term newborns who survived at most 12 days were examined to study thyroid development in late intrauterine life. Total iodine thyroglobulin (Tg), T4, T3 and rT3 were assayed before and after hydrolysis with pronase. In the same maternity unit the cord-serum of 25 healthy term newborns was also assayed and the urine iodine on the day of delivery was tested in 52 newborns and their mothers. Results are expressed as mean values +/- SD. The thyroid gland weight ratio to body weight for the preterms was 0.063 +/- 0.024; thyroid Tg content 5.9 +/- 4.0 mg/g; total thyroid iodine 41.7 +/- 35.1 micrograms/g; Tg iodination 0.72 +/- 0.37%; hormone quota of Tg iodine 39.9 +/- 12.4. Molar ratios were: T4/Tg 3.3 +/- 1.6, T3/Tg 0.25 +/- 0.14, rT3/Tg 0.072 +/- 0.059. Considerable differences in thyroid iodine content and Tg iodination were observed although the availability of iodine was presumably the same. The high Tg iodination reflects the enhanced uptake of the fetal thyroid. The quantity of iodine involved in hormonogenesis varied greatly among fetuses of the same gestational age, this being in the third term as active as in fully mature thyroids. Hormonogenesis seemed preferentially directed to production of T4 and rT3, which, in the latter case, is higher peripherically. Four preterms with a particularly low level of hormonogenesis were not seen to have deficient levels of thyroid iodine or Tg iodination and the most compromised step was the iodine utilization in the production of hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital Hypothyroidism↗
PubMed1984

Thiocyanates and iodine in endemic goiter in Italy.

Urinary iodine (I) and serum and urinary thiocyanates (SCN-) were determined in sample groups from 12 areas of endemic goiter in Italy. The mean urinary I level of 637 subjects with thyroid 0 was 67 +/- 31 (mean +/- SD) microgram/liter, that of 648 with goiter 54 +/- 29 micrograms/liter. Mean serum and urinary SCN- were 2.44 +/- 1.36 mg/liter and 2.58 +/- 1.36 mg/liter in 887 and 1531 subjects, respectively. The thiocyanates data enabled a distinction to be drawn between two groups (populations). Population I included 73% of the adults and 92% of the school-children (6-16 yr). Its thiocyanate values were logarithmically spread around means of 1.24 +/- 0.6 mg/liter (serum) and 1.24 +/- 0.57 mg/liter (urine), whereas those of population II were widely dispersed around means of 6.1 +/- 3.0 mg/liter and 8.08 +/- 5.5 mg/liter respectively. The boundary between the two populations was set at 3 mg/liter urine SCN-. This distinction was substantiated by the fact that 90% of those in population II smoked 10 or more cigarettes a day, whereas population I comprised occasionally smokers only. It is believed that only the values in population I can be regarded as representative of thiocyanates either endogenous or due to dietary dietary intake: these values never differed more than 60% regardless of the areas, seasons, or dietary habits. As urinary SCN- levels rose, there was also an increase in urinary iodine excretion within certain limits. This, however, did not interfere with thyroid secretion. The urinary I/SCN- ratio was lower in subjects with goiter. This was because their iodine levels were lower, whereas SCN- values were much the same in subjects with and without goiter. We have found no correlation between thiocyanate itself and goiter.

Adolescent↗