Specific Two-site Enzyme Immunoassay Research Articles

Metrifonate, like acetylcholine, up-regulates neurotrophic activity of cultured rat astrocytes

BackgroundMetrifonate is an inhibitor of acetylcholinesterase (AChE). Several studies confirmed its positive effects on cognitive impairment in Alzheimer's disease but it was due to adverse events withdrawn from clinical trials. Based on the importance of astrocytes in physiological and pathological brain activities we investigated the impact of metrifonate and, for comparison, acetylcholine on intrinsic neurotrophic activity in these cells. MethodsMetabolic activity, intracellular adenosine 5′-triphosphate (ATP) levels and lactate dehydrogenase (LDH) release was measured to examine the impact of metrifonate on viability and integrity of cultured rat cortical astrocytes. The influence of metrifonate, acetylcholine and selective cholinergic ligands on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) synthesis and secretion was determined by specific two-site enzyme immunoassays. ResultsExposure of cultured astrocytes to metrifonate displayed no toxic effects on cell viability. Metrifonate and acetylcholine potently and transiently elevated NGF and BDNF, but not NT-3, protein levels and secretion with different intensity and time frame of their maximal response. Stimulatory effect on NGF was mimicked by selective nicotinic receptor agonist nicotine and completely blocked by nicotinic antagonist mecamylamine. The impact on BDNF synthesis was mimicked by muscarinic receptor agonist pilocarpine and abolished by selective muscarinic antagonist scopolamine. ConclusionsMetrifonate up-regulates astrocytic NGF and BDNF synthesis in the same manner as acetylcholine, their effect depends on different cholinergic pathways. These results suggest a trophic role of metrifonate, based on a well-known neurotrophic activity of NGF and BDNF in vivo.

Umbilical activin A concentration as an early marker of perinatal hypoxia

Objective: To evaluate activin A as a potential, early marker of perinatal hypoxia and to analyze factors, other than hypoxia, which influence on activin A concentration. Methods: Umbilical cord blood samples were collected from 86 newborns with gestational age 30–41. Of the 86 newborns, 26 were regarded as hypoxic. Activin A concentrations were measured by means of specific two-site enzyme immunoassays. Activin A concentrations were correlated with newborns’ gender, week gestation, mode of delivery and blood gas measurements. Results: Activin A levels were significantly higher in hypoxic than nonhypoxic newborns (medians, minimum and maximum values: 1.516; 0.149 –1.974 versus 0.368; 0.054 – 1.041 ng/mL, p = 0.0452). Activin A concentration was significantly higher in male newborns (p = 0.0074). Activin A levels were lower in term than preterm babies but the differences were not statistically significant (p = 0.2368 in hypoxic, p = 0.2487 nonhypoxic). Mode of delivery did not influence on activin A concentration (p = 0.8293 hypoxic, p = 0.9458 nonhypoxic). The differences of occurrence of intraventricular hemorrhage (IVH) in both group was not statistically significant (p = 0.61). Conclusions: Umbilical artery activin A combined with other markers of hypoxia could be a useful marker of perinatal hypoxia. Concentration of activin A is significantly higher in male newborns. The mode and time of delivery have no influence on activin A concentration.

High concentrations of activin A in the peritoneal fluid of women with epithelial ovarian cancer.

The aim of the present study was to evaluate the concentrations of activin A in the peritoneal fluid of women with epithelial (serous) ovarian cancer. A group of 160 women was studied and divided in four subgroups as follows: 1) serous ovarian carcinoma (n = 32); 2) serous ovarian cystadenoma (n = 20); 3) endometriosis (n = 53); and 4) healthy controls (n = 55), including both fertile (n = 32) and postmenopausal women (n = 23). Specimens of peritoneal fluid were collected during surgical interventions, and activin A was quantified using a specific two-site enzyme immunoassay. Peritoneal fluid activin A concentrations in women with ovarian carcinoma were about five-fold higher than those found in the control group (median [interquartile range] = 7.60 [2.85-10.15] and 1.50 [1.00-2.50] ng/mL, respectively, P <.001). In contrast, the women with benign serous cystadenoma had peritoneal fluid activin A concentrations (1.50 [1.0-2.70] ng/mL) similar to those of the control group. High peritoneal fluid activin A levels (>2 multiples of the mean) distinguished carcinoma from cystadenoma with a sensitivity of 72% and a specificity of 80%. The follow-up of nine patients with stage IIIc ovarian cancer showed no apparent relationship between the peritoneal fluid activin A levels and overall survival. No significant difference in peritoneal fluid activin A concentrations between patients with endometriosis and control women was observed. Most women with serous ovarian carcinoma had high concentrations of activin A in the peritoneal fluid, supporting a possible role of this growth factor in ovarian cancer.

Sensitive immunoassays for human and rat GMFB and GMFG, tissue distribution and age-related changes.

We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21-30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat. The present EIA systems are sufficiently sensitive for studying GMFs in human and rat organs.

Nerve growth factor serum concentrations in healthy human volunteers: physiological variance and stability

Human nerve growth factor (NGF) serum concentrations were measured in a healthy sample of 126 participants by a modified highly sensitive and specific two-site enzyme immunoassay. The measured NGF concentrations differ considerably from a normal distribution. The median NGF concentration was 19.68 pg/ml with an interquartile range of 11.06–41.74 pg/ml, which means that 50% of the NGF levels are in this range. In our healthy sample, we found no gender differences but a slight age-related decrease of NGF ( r=−0.1326, P=0.1560). Moreover intraindividual stability of NGF was examined in ten volunteers, where no significant changes of serum NGF concentrations were detected over 4 weeks. This stability of our repetitive measurements over 4 weeks suggests that this neurotrophin may be an intraindividually solid marker at least in human serum.

Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans.

Metastin is a novel peptide that was recently isolated from human placenta as the endogenous ligand of an orphan heptahelical receptor, hOT7T175. Metastin has been shown to suppress the motility of hOT7T175-transfected melanoma cells; however, studies of the physiological function of metastin have begun only recently. To investigate the possibility that metastin is an endocrine peptide, we determined the immunoreactive (ir-) metastin concentration in human plasma using our newly developed, sensitive, and specific two-site enzyme immunoassay. The plasma concentrations of ir-metastin in males and females were 1.30 +/- 0.14 (n = 12) and 1.31 +/- 0.37 fmol/ml (n = 10), respectively. As metastin is known to be abundant in human placenta, the ir-metastin concentration in the maternal plasma was then determined. The ir-metastin concentrations were 1230 +/- 346 fmol/ml (n = 11) in the first trimester, 4590 +/- 555 (n = 16) in the second trimester, and 9590 +/- 1640 (n = 12) in the third trimester. On d 5 after delivery, the ir-metastin concentration returned to nearly the nonpregnant level (7.63 +/- 1.33 fmol/ml; n = 10), suggesting that ir-metastin increases in pregnancy and is derived mainly from the placenta. The plasma from both nonpregnant and pregnant women showed a single ir-metastin peak at the same retention time as authentic metastin on reverse phase HPLC analysis, indicating that the major portion of the circulating metastin, as determined by our two-site enzyme immunoassay, represents endogenous metastin. Histochemical studies of human placenta localized metastin mRNA and immunoreactivity to the syncytiotrophoblasts. The present study provides evidence for metastin as a novel placenta-derived hormone in humans.

Corticotropin-releasing Factor, Urocortin and Endothelin-1 Stimulate Activin A Release from Cultured Human Placental Cells

Human placenta produces activin A, a glycoprotein belonging to the transforming growth factor beta superfamily, which modulates several placental immune and endocrine functions. However, substances involved in controlling placental activin A production are not yet completely elucidated. The aim of the present study was to investigate the effects of placental products, corticotropin-releasing factor (CRF), urocortin, prostaglandin E2 (PGE2) and endothelin-1 (ET-1) on activin A release from cultured human placental cells. Placental tissue was collected at term from normal pregnancies and a trophoblast-enriched cell preparation was cultured for 48h. The test substances were applied (concentration from 10−9–10−7M) and the medium was harvested after 3h incubation; vehicle-treated cells (controls) were present in each experiment. Activin A concentrations in culture medium were measured by using a specific two-site enzyme immunoassay. The addition of CRF resulted in a dose-related increase of activin A concentrations (P< 0.01). The stimulatory effect of CRF was significantly reversed by α-helical CRF9-41, the CRF receptor antagonist. Urocortin showed a stimulating effect on activin A release from placental cells (P< 0.05) but not dose-related; the effect of urocortin was reversed by an equimolar dose of CRF antagonist, astressin. ET-1 significantly increased activin A concentrations in the culture medium only at the highest concentration, 10−7M (P< 0.05). No difference in activin A release was observed after incubating the cells with PGE2. The evidence that CRF, urocortin and ET-1 stimulate activin A secretion from cultured placental cells suggests that these vasoactive factors may affect the changes of placental activin A secretion in pre-eclamptic woman.

Serum activin A and follistatin in disorders of spermatogenesis in men.

Inhibin, activin and follistatin are glycoprotein hormones produced by the gonads. Recent studies have shown that inhibin B is the predominant form of inhibin in the circulation in men. The objective of this study was to investigate circulating levels of activin A and follistatin in disorders of spermatogenesis in men and their relationship with FSH and inhibin B. Serum from five different groups of men was prospectively collected and stored at -20 degrees C. The groups were men with: (i) proven fertility (controls) (n=20), (ii) primary testicular failure (n=15), (iii) obstructive azoospermia (n=10), (iv) oligospermia (n=10) and (v) miscellaneous sperm dysfunction (n=40). WHO criteria (1992) were used for semen characterisation. Serum concentrations of 'total' activin A, follistatin, FSH and inhibin B were measured using specific two-site enzyme immunoassays. Activin A levels were significantly lower than in the controls in the obstructive azoospermia group and higher in the miscellaneous sperm dysfunction group. Serum follistatin levels did not significantly vary in any group compared with the controls. Circulating levels of FSH were higher than in the controls in the primary testicular failure and obstructive azoospermic group. Levels of inhibin B were lower than in the controls in all disorders of spermatogenesis studied. This study demonstrates that activin A and follistatin are in the circulation in males and activin A levels are significantly lower in obstructive azoospermia and higher in miscellaneous sperm dysfunction than in controls. The mechanism involved in altering the levels of activin A in these conditions is not clear. However, high follistatin:activin A molar ratios (>2.5) in all groups suggests that all activin A in the circulation is bound to follistatin in males.

Human mammary gland and breast carcinoma contain immunoreactive inhibin/activin subunits: evidence for a secretion into cystic fluid.

Inhibins and activins are members of the transforming growth factor beta superfamily and are known to modulate the growth and differentiation of several cell types. The present study investigated the localization of inhibin and activin subunits in human normal and pathological breast tissues. A cross-sectional study comparing the expression of inhibin/activin subunits alpha, betaA and betaB in surgical specimens from women undergoing reductive mammoplasty (classified, according to the phase of the menstrual cycle, as follicular, luteal, or postmenopausal), and patients submitted to lumpectomy for fibrocystic disease, benign (intraductal papilloma, adenomyoepithelioma, and hamartoma) or malignant breast neoplams (intraductal, intralobular, and invasive carcinoma). Immunohistochemistry was used to localize inhibin alpha and activin betaA and betaB subunits in the cytoplasm of epithelial cells of mammary glands. Dimeric activin A, inhibin A and inhibin B were measured by specific two-site enzyme immunoassay in the cystic fluid collected from patients with fibrocystic disease. An intense staining for the alpha inhibin subunit and a mild staining for betaA and betaB subunits were present in samples obtained from normal breast tissue regardless of menstrual cycle phase, and in fibrocystic disease and benign neoplasms. Carcinoma cells stained weakly to moderately for alpha subunit and were negative for betaA and betaB subunits. Fibrocystic disease was associated with absence of betaA subunit expression in normal epithelial cells and intense staining for all subunits in the apocrine cells. Immunoreactive inhibin A, inhibin B, and activin A were also present in cystic fluid, suggesting a local secretion of these proteins. These data suggest a local expression and secretion of inhibin and activin in human normal, fibrocystic disease and neoplastic breast tissues. The low expression of these proteins may facilitate abnormal cell proliferation in breast carcinoma.

Distribution and Characterization of Immunoreactive Prolactin-Releasing Peptide (PrRP) in Rat Tissue and Plasma

We established a sensitive and specific two-site enzyme immunoassay (EIA) for prolactin-releasing peptide (PrRP) using two region-specific monoclonal antibodies. We investigated the tissue distribution and the plasma concentration of immunoreactive (ir-) PrRP in rats using this assay. Ir-PrRP was widely distributed in the central nervous system and pituitary gland. The highest concentration of ir-PrRP was found in the hypothalamus. In peripheral tissues, appreciable levels of ir-PrRP were found only in the adrenal gland. The mean plasma concentration of ir-PrRP was 0.13 ± 0.01 fmol/ml (mean ± SEM). In reverse-phase and gel-filtration high performance liquid chromatography, hypothalamic ir-PrRP eluted at a position identical to that of PrRP31 and PrRP20. On the other hand, ir-PrRP from the adrenal gland and plasma eluted only at the position of synthetic PrRP31, indicating that molecular forms of ir-PrRPin vivodiffered among tissues.

Development of a highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34): application to pharmacokinetic study on intranasal parathyroid hormone (1-34) in human.

A highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34) (PTH(1-34)) and its usability for the pharmacokinetic study are described. Plasma samples were incubated simultaneously with 2,4-dinitrophenylated anti-PTH(1-34) IgG and anti-PTH(1-34) Fab'-beta-D-galactosidase conjugate. The immune complex formed of the three components was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the polystyrene balls was assayed by fluorometry. The practical detection limit of PTH(1-34) was 50 fg (12 amol)/0.05 ml of sample and 1 pg/ml as the concentration and practically no interference occurred by PTH(1-84) and PTH-related protein (1-34) up to 300 pg/ml and 10 ng/ml, respectively. The application of this method has enabled us to directly estimate the bioavailability of PTH(1-34) dosed intranasally at the prescribed level (0.090 mg). The pharmacokinetic parameters of the intranasal PTH(1-34) (n = 4) thus estimated were as follows: the area under the plasma concentration-time curve (AUC) = 20,500+/-15,900(SD) pg.min/ml; the mean residence time (MRT) = 194+/-16.3(SD) min; and the maximal concentration (Cmax) = 98+/-51 (SD) pg/ml with the maximal time (Tmax) = 35.0+/-12.2(SD) min.

Establishment of Monoclonal Antibodies against Human Nerve Growth Factor

We have generated and characterized a monoclonal antibody to human nerve growth factor (hNGF). The monoclonal antibody NGFA-133 neutralizes hNGF activity, as assayed by neurite-outgrowth of nerve cells from chick embryonal dorsal root ganglion. Using this antibody, we have developed a sensitive and specific two-site enzyme immunoassay (EIA) system for hNGF. The assay is based on a sandwiching of the antigen between NGFA-133 coated on a microtiter plate and the same monoclonal antibody (NGFA-133) conjugated with horseradish peroxidase (HRP). The two-site EIA was sensitive enough to detect 920 fg/well of hNGF and did not cross-react either human neurotrophin-3 (hNT-3) or sodium dodecyl sulfate (SDS) denatured hNGF.

A Highly Sensitive and Specific Sandwich Enzyme Immunoassay for Detection of Human Chorionic Gonadotropin in Trophoblastic Disease

A highly sensitive and specific two-site enzyme immunoassay (EIA) kit for human chorionic gonadotropin (hCG) has been examined and applied to the detection of hormones in the serum of normal individuals and patients with trophoblastic disease. This EIA uses a solid phase coupled antibody to the unique COOH terminal peptide region of the hCG beta-subunit (beta-CTP) and an enzyme conjugated monoclonal antibody to the hCG beta-subunit. The overnight 2 step assay measured as little as 0.2 mIU/ml serum without hLH interference and a rapid one step assay was newly developed in our laboratory. Assays of serum from normal men and nonpregnant women of reproductive age indicated that most individuals did not have detectable serum levels of hCG immunoreactivity, although a minority had minute amounts. In contrast, women with non-functional ovaries had detectable levels of hCG immunoreactivity with a mean value of 0.86 mIU/ml serum. The extremely high sensitivity and specificity of the EIA in detecting hCG provides substantial advantages for the detection of early pregnancy and for monitoring trophoblastic tumors.

The Gene Encoding Nerve Growth Factor Is Expressed in the Immature Rat Ovary: Effect of Denervation and Hormonal Treatment*

The rat ovary is innervated by sympathetic nerve fibers. Since the development and survival of peripheral sympathetic neurons innervating nonreproductive organs have been shown to depend on the production of nerve growth factor (NGF) by the innervated tissues, the present experiments were undertaken to determine if the immature rat ovary has the capability of synthesizing NGF. Blot hybridization of ovarian polyadenylated RNA (A+-RNA) to a NGF cRNA probe revealed the presence of a 1.3- to 1.4-kilobase (kb) mRNA species similar to mature NGF mRNA detected in mouse submaxillary gland, a source rich in NGF. Quantitation of NGF protein by a sensitive and specific two-site enzyme immunoassay demonstrated the presence of NGF in juvenile ovaries at levels comparable to those found in other sympathetically innervated tissues. Neither denervation of the ovary nor treatment with gonadotropins (hCG and FSH) or somatomammotropins (PRL and GH) affected the levels of NGF mRNA. However, denervation significantly increased NGF levels, suggesting that, as in other target tissues, denervation prevents the retrograde transport of NGF by the sympathetic terminals and leads to accumulation of the protein at its site of production. It is concluded that 1) the developing ovary is able to both transcribe the NGF gene and translate its mRNA into NGF protein; and 2) the NGF content in the ovary is regulated by its innervation. The results provide the biochemical basis for the concept, elaborated in the companion paper, that NGF through its trophic actions on ovarian sympathetic neurons contributes to the regulation of ovarian development and, hence, to the acquisition of female reproductive capacity.