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M Pazzagli

Publications and source records attributed to M Pazzagli.

At least 55 records · Page 3Linked to original sources
PubMed1987

Chemiluminescence immunoassay for somatomedin C in serum.

To select the best tracer for use in a competitive immunoassay, we conjugated human somatomedin C (SmC) to various chemiluminescent compounds via two different synthetic pathways. Naphthylhydrazides and arylhydrazides, used as the labels, were incorporated via their imidate or their succinimide esters. Conjugating the carboxy terminal of (amino ethyl)ethyl-isoluminol to SmC via a succinimide linkage supplied the most sensitive detection limit and the most immunoreactive conjugate. We developed an immunoassay based on the use of this conjugate, and evaluated dextran-coated charcoal, second-antibody precipitation, and solid-phase immunoprecipitation for separating bound and free label. This chemiluminescent method has a detection limit of 16 pg per tube, and it is accurate and precise. Correlation studies with a conventional radioimmunoassay (x) for SmC gave the following regression equation: y = 0.66x + 3.76 (r = 0.953, n = 30); the slight discrepancies between the two methods are probably ascribable to the use of different antibodies. We thus propose this chemiluminescence immunoassay as an inexpensive and sensitive alternative to radioimmunoassay for measuring SmC in serum or in extracts of serum.

DNA, Recombinant↗
PubMed1986

Measurement of testosterone and its 5-alfa-reduced metabolites in human prostatic tissue using isotope dilution mass spectrometry.

5 alpha-Dihydrotestosterone (DHT) has been widely measured in human prostatic tissue using RIA since it is found to be involved in pathogenesis of human prostatic hyperplasia (BHP) and to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC/MS method for the simultaneous determination of testosterone (T), dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstan-3 beta-17 beta-diol (3 beta-diol) in prostatic tissue was developed based on the isotopic dilution technique. Trideuterated internal standards of each compound were previously synthesized in our laboratory. After previous extraction and purification on Sep-Pak C-18 cartridges and Lipidex DEAP columns, T and its metabolites were measured as heptafluorobutyric esters (HFB). Quantitative analysis was performed on a VG 7070 EQ mass spectrometer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (means +/- SD; ng/g tissue) found in 9 human hypertrophic prostates were: T, 0.71 +/- 0.43; DHT, 4.46 +/- 1.41; 3 alpha-diol, 0.34 +/- 0.23; 3 beta-diol, 0.14 +/- 0.32.

Androstane-3,17-diol↗
PubMed1986

Measurement of transferrin in human seminal plasma by a chemiluminescent method.

We describe a luminescence immunoassay for measuring transferrin in human seminal plasma. Human transferrin conjugated with 7-[(N-4-aminobutyl)-N-ethylamino]naphthalene-1,2-dicarboxylic acid hydrazide was used to monitor the immunological reaction. The conjugate was stable for at least one year. The sensitivity (2 ng per tube) of the assay allows measurement of this protein in diluted seminal plasma. Results by this method (y) correlated well (r = .9681) with those by a conventional RIA method (x): y = 1.000x + 1.646 micrograms per ejaculate. Seminal transferrin concentrations are reported for normal control subjects, vasectomized subjects, and infertile patients. The method described appears suitable for measurement of seminal transferrin as an index of Sertoli cell function in male infertility.

Humans↗
PubMed1985

Pregnanediol-3 alpha-glucuronide measured in diluted urine by mass spectrometry with fast atom bombardment/negative-ion ionization.

We describe a mass-spectrometric method based on the fast atom bombardment ionization technique in the negative-ion mode for measuring pregnanediol-3 alpha-glucuronide in diluted urine from women. The procedure requires addition of testosterone-17 beta-D-glucuronide (2.5 micrograms/25 microL) to the urine sample as internal standard, and the sample is added directly to the fast atom bombardment target with no further manipulation. We have assessed and evaluated the method by the traditional criteria of reliability.

Adult↗
PubMed1984

Homogeneous luminescence immunoassay for total estrogens in urine.

We describe an homogeneous luminescence immunoassay for "total" estrogens in enzymically hydrolyzed urine from nonpregnant women. The antiserum, raised against estriol-16,17- dihemisuccinate conjugated to bovine serum albumin, specifically bound the C-19 steroids carrying the estrogen-characteristic phenolic group. 17 beta-Estradiol conjugated with aminobutylethyl isoluminol was used to monitor the immunological reaction; this conjugate was stable for at least two years. Because binding to the antiserum markedly enhances the light-producing efficiency of the tracer, no separation of bound and free antigen is necessary. Results (microgram/24 h) by this method (y) correlated well (r = 0.958) with those by a conventional fluorometric (x) method (y = 2. 51x - 2.83). The sensitivity (detection limit) is 4 micrograms/L and the precision compares well with that of commonly used RIA methods. The method appears suited to large numbers of samples, as in menstrual cycle monitoring.

Cross Reactions↗
PubMed1984

On-line computer analysis of chemiluminescent reactions, with application to a luminescent immunoassay for free cortisol in urine.

We interfaced a microcomputer on-line with a luminometer to acquire the light signal of chemiluminescent reactions from a photomultiplier and then compute significant parameters of light emission and kinetic "shape" indices. Using this system to study interferences from biological samples on the measurement of chemiluminescent reactions, we observed that such effects are usually associated with modifications of the shape of the light-emission kinetics. These results suggest that a simultaneous evaluation of the shape of a chemiluminescent reaction and the measurement of light emission can be combined to assess luminescent immunoassays as an internal control of the interferences in measurements of the chemiluminescent tracer. As an example of this approach, we developed and validated a luminescent immunoassay for free cortisol in diluted urine. Dextran-coated charcoal is used for bound-free separation.

Chemical Phenomena↗
PubMed1983

A solid phase chemiluminescent immunoassay (LIA) for testosterone glicuronide in diluted urines.

A chemiluminescent immunoassay (LIA) method in solid phase for the measurement of testosterone 17 beta-D-glicuronide (TG) in diluted urine is described, which utilizes as tracer a TG-isoluminol conjugate (TG-ABEI). An IgG fraction of antiserum of TG-BSA, has been passively adsorbed to the walls of polystyrene tubes. After the binding reaction the coated tubes were washed with buffer and the measure of chemiluminescence reaction was performed at high pH. The assay was validated in terms of specificity, accuracy, sensitivity and precision. The values obtained by chemiluminescence immunoassay were compared with that obtained by the RIA method, and the two methods agreed well (r = 0.95, n = 28). The assay method offers the advantage of speed and does not involve the use of radioisotopes or of a centrifugation step. Preliminary results show that the mean 24 h urinary TG excretion in a group of hirsute women is higher than in the control group, and decreases after suppression with dexamethasone for 1 month of therapy.

Cushing Syndrome↗
PubMed1983

Preparation and evaluation of steroid chemiluminescent tracers.

Six different isoluminol derivatives have been synthesized and evaluated as potential labels for conjugation to steroid molecules. The resulting steroid-chemiluminescent tracers have been investigated in terms of detection limit and affinity for the homologous antibody. The conjugation to the steroid molecule did not significantly affect the quantum yield of the chemiluminescent label. Consequently the detection limit was very similar using tracers with the same chemiluminescent label and different steroids and it was always at picomolar levels. The affinity of the chemiluminescent tracer versus the homologous antibody was evaluated using tritiated steroids as reference. The bridge and the chemiluminescent molecule could influence the affinity of the chemiluminescent tracer which could be higher, similar or lower than that of the tritiated steroid. In conclusion some isoluminol derivatives (i.e. ABEI or AEEI) can be considered as universal labelling compounds for steroids and their metabolites, because they possess a high light efficiency (similar to luminal itself) and favourable reaction conditions. Moreover the conjugation to the steroid molecule does not modify their chemiluminescent properties.

Animals↗
PubMed1981

Evaluation of different progesterone-isoluminol conjugates for chemiluminescence immunoassay.

The effect of varying the length of the alkyl bridge linking the chemiluminescent label isoluminol to progesterone on the light yield and binding affinity of progesterone chemiluminescent-marker conjugates was investigated. For this purpose five different derivatives of isoluminol, aminoethyl isoluminol (AEI), aminoethylethyl isoluminol (AEEI), aminobutyl isoluminol (ABI), aminobutyl-ethyl isoluminol (ABEI) and aminoethyl-ethyl isoluminol (AHEI) were covalently linked through a peptide bond to progesterone-11 alpha -hemisuccinate (P-11-HS). The resulting progesterone chemiluminescent-marker conjugates were then evaluated as potential labels for the development of an immunoassay based on monitoring chemiluminescence. These conjugates were able to compete with tritiated progesterone for the binding sites of an antibody raised against progesterone-11 alpha-HS bovine serum albumin, and they showed higher affinity than unaltered progesterone. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant, catalyst and conjugate design. Under optimal conditions, all the conjugates were detectable at femtomolar levels. The lowest detection limit was obtained using P-11-HS-ABEI (0.1 fmol). These results indicated that immunoassay techniques based on chemiluminescence can be developed with these labels.

Animals↗
PubMed1981

Luminescent immunoassay (LIA) for progesterone in a heterogeneous system.

An immunoassay procedure for determination of progesterone in human plasma is described which utilizes chemiluminescence as the endpoint. The assay utilized progesterone-11-hemisuccinate-aminobutyl-ethyl-isoluminol (P-11-HS-ABEI) as the chemiluminescent marker conjugate and dextran coated charcoal for the separation of bound and free fractions of the ligand. An assay procedure for progesterone was established and validated in terms of sensitivity and precision, and assay results were compared with radioimmunoassay, using tritiated progesterone as the tracer. The two methods agreed well (n=35, r=0.96). The most important advantage of this assay is the elimination of problems inherent in the use of radioactive materials.

Azides↗
PubMed1981

Spermatic and peripheral oestradiol levels in patients affected by azoospermia due to seminiferous tubular damage.

Plasma levels of testosterone, androstenedione and oestradiol were determined in the spermatic venous blood of both testes of 17 patient affected by azoospermia due to tubular damage (Group I). The results were compared with those found in 5 patients affected by azoospermia of obstructive origin and 5 patients with an inguinal hernia (Group II). Mean spermatic levels of testosterone and androstenedione were not significantly different in the two groups, while the mean (+/- SE) oestradiol spermatic level was significantly higher in patients of Group I (5.02 +/- 0.75 nM/l vs. 2.20 +/- 0.365 nM/l; P less than 0.05). Moreover, while the testosterone/androstenedione and the androstenedione/oestradiol ratios were not significantly different in the two groups, the mean (+/- SE) testosterone/oestradiol ratio was significantly lower in patients of Group I (552.71 +/- 80.94 vs. 939.86 +/- 129.45; P less than 0.025). Peripheral testosterone and androstenedione mean levels were not significantly different between the two groups while the mean peripheral oestradiol level (+/- SE) was significantly higher in Group I (0.107 +/- 0.021 nM/l vs. 0.038 +/- 0.05 nM/l; P less than 0.025). Peripheral oestradiol was not significantly related to peripheral FSH, nor to spermatic oestradiol in both groups. These results suggest the possibility that oestradiol may be involved in the pathogenesis of some cases of male infertility.

Adult↗
PubMed1980

An immunoassay for plasma cortisol based on chemiluminescence.

An immunoassay procedure for the determination of cortisol in human plasma is described, which utilizes chemiluminescence as the end point. A cortisol-isoluminol conjugate serves as the chemiluminescent marker. The light emission by this conjugate upon oxidation is delayed by prior incubation with anti-cortisol IgG, but not by unrelated gamma-globulin. This delayed light emission was inhibited by cortisol in a dose-dependent manner, with a linear range of 20-1000 pg steroid/assay tube. A competitive protein binding assay based on this procedure was applied to methylene chloride extracts of cortisol from normal and pathological human plasma (2-40 micrograms/100 ml). Cortisol values obtained by this procedure agreed well with those obtained by radioimmunoassay, using the same antiserum with tritiated cortisol as the label (r = 0.98). The chemiluminescence immunoassay is comparable to radioimmunoassay with regard to sensitivity, specificity, precision and accuracy. The advantage of the new assay procedure is that it obviates the need for counting radioactivity and for separation of bound and free ligand.

Humans↗