Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum.

4,4'-Isopropylidenediphenol, bisphenol A (BPA), was derivatized to BPA-carboxymethylether (BPA-CME), BPA-carboxypropylether (BPA-CPE) and BPA-carboxybutylether (BPA-CBE), and then linked to bovine serum albumin (BSA). The BPA-BSA conjugates were injected into female New Zealand White rabbits, which then generated six kinds of polyclonal antibodies. In addition, BPA and bisphenol B (BPB)-enzyme conjugates were derivatized to BPA-CME, BPA-CPE, BPA-CBE, BPA-carboxyphenylether (CPhE) and BPB-CPE, and then linked to horseradish peroxidase (HRP). An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of BPA standards. Although anti-BPA antibodies cross-reacted with BPB by more than 13.6% at all dilutions used, cross-reaction with phthalates and phenols occurred only less than 0.1%. The combination with the highest sensitivity was obtained using anti-BPA-CME-BSA antibody and BPA-CPhE-HRP conjugate. ELISA successfully detected BPA in human serum at concentrations as low as 0.3 ng mL(-1), and over a measurable range of 0.3-100 ng mL(-1). Recovery tests were carried out by adding BPA to three kinds of human serum, and ranged from 89.7 to 97.3%, from 85.4 to 94.9% and from 81.9 to 97.4%, respectively. The correlation between the results from ELISA and gas chromatography-mass spectrometry (GC-MS) for BPA in spiked serum was r2 = 0.990, indicating that the proposed method is a potential tool for screening a large number of human serum samples.

Plasma calcium and calcitonin levels in eels fed a high calcium solution or transferred to seawater.

To examine the physiological role of calcitonin (CT) in calcium homeostasis of teleosts, we compared calcium and CT levels in freshwater eels fed a high calcium-consomme solution (Ca2+: 1.25 M; 1 ml/100 g body wt) into the stomach (Experiment I), and in freshwater eels transferred from freshwater to seawater (Experiment II). In experiment I, plasma calcium and CT levels in the high calcium-treated eels rapidly increased (calcium: 2.63 mM at 0 h to 8. 50 mM at 3 h; CT: below detection level at 0 h to 1118.2 pg/ml at 3 h). Plasma calcium and CT levels in the control eels remained below detection level during the 3 h of the experiment. In experiment II, the plasma CT levels did not increase, although the plasma calcium levels increased from 3.23 mM at 0 h to 4.10 mM at 8 h. Therefore, in eels, we demonstrate a correlation between plasma CT and plasma calcium raised by dietary calcium in the consomme form, but it does not participate in the initial processes of seawater adaptation.

Calcitonin cells in the intestine of goldfish and a comparison of the number of cells among saline-fed, soup-fed, or high Ca soup-fed fishes.

Calcitonin-immunoreactive cells were found in the intestine of goldfish. These cells were distributed mainly in the anterior part of the intestine, dispersed in the intestinal epithelium. The nucleus was located in the basal portion of the serosal side, and the cytoplasm was elongated to the luminal side. From the anterior part of the intestine, cDNA fragments with the same nucleotide sequence as that of the goldfish calcitonin gene were amplified by RT-PCR method. After administration of one of three kinds of solutions (saline, consommé soup, or high Ca consommé soup) into the digestive tract of the goldfish, the number of those cells was the largest in the consommé group at 6 h after ingestion, although blood Ca levels were the highest in the high Ca consommé group. The function of calcitonin cells in the intestine may be to restrain the acute absorption of nutrients and not to control blood Ca levels.

Development of a time-resolved fluoroimmunoassay (TR-FIA) for testosterone: measurement of serum testosterone concentrations after testosterone treatment in the rainbow trout (Oncorhynchus mykiss).

A sensitive time-resolved fluoroimmunoassay (TR-FIA) for testosterone was developed, and the assay system was used for measuring serum testosterone concentrations in rainbow trout. Testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (T-3-CMO-BSA) was immobilized by physical adsorption to the wells of microtiter plates. A competitive assay using two antibodies was performed among T-3-CMO-BSA in the solid-phase, unknown amounts of testosterone, testosterone antibodies, and europium labeled secondary antibodies, followed by measurements using a time-resolved fluorometer (DELFIA system). The TR-FIA had a sensitivity of 0.075 pg/50 microliters sample (1.5 pg/ml), and the range of the assay system was between 1.5 pg/ml and 25 ng/ml. The intra- and interassay coefficients of variation for the testosterone TR-FIA were satisfactorily low, and were between 1.62 and 6.38% and 2.96 and 8.29%, respectively. The assay system was applied to measure the serum testosterone concentrations after an injection of testosterone dissolved in saline, propyleneglycol, or coconut oil. Among the three solvents, the coconut oil group showed continuously high serum testosterone level. In contrast, the saline and propyleneglycol groups had maximum concentrations 24 hr after the injection, but their levels were significantly lower than that of the coconut oil group. The testosterone TR-FIA method is sensitive, repeatable, and is as accurate as conventional RIAs. It is very good for measuring serum testosterone concentrations.

[Enzyme labeling methods and it's specificities].

Enzyme labeled antigen for use in ELISA of Hapten (steroids, prostanoid, carbohydrate, nucleic acid, peptide, herbicide, insecticide and antibiotic) have usually been prepared by condensation of carboxy group of hapten with amino groups of lysine residue in enzyme. The horseradish peroxidase (HRP) is best suitable as labeling enzyme, therefore it is small molecular, and substrate turnover is much higher compared to the other enzymes. The mixed anhydride and carbodiimide methods have mainly been used the preparation of hapten conjugate BSA, but not satisfactory for enzyme labeling. The N-hydroxysuccinimide ester (NHS: active ester) method is satisfactory with respect to reproducibility and sensitivity. The sensitivity is related to the bridging phenomenon: One of the disadvantages of the homologous labels is that the antibody shows an affinity not only for the Hapten but also for the bridge which connects the Hapten to carrier protein. We have developed a sensitive bridge heterologous EIA for progesterone (P) using geometrical isomers of P-3 (E/Z) (O-carboxymethyl) oxime-N-hydroxysuccinimide esters [ef Ab of P-3 (E) CMO-BSA/P-3 (Z) CMO-HRP]3). The sensitivity of heterologous proved to be higher than a homologous EIA or a conventional RIA. It seem like that a 1:1 steroid-enzyme conjugate is suitable for obtaining a high sensitivity. The avidin-biotin (AB) system provides great versatility, since by conjugation with an appropriate label, the AB assay can be used with any chosen detector. Furthermore, IgG can be labels with biotin without significantly influencing their immunological activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent coupling of a steroid to microwell plates for use in a competitive enzyme-linked immunosorbent assay.

The covalent coupling of a model steroid, 17 alpha-hydroxypregnenolone, to the wells of the microtiter plate, CovaLink NH, for use in a competitive enzyme-linked immunosorbent assay is described. This plate has secondary amino groups bound to its surface. A carboxylated derivative of the steroid was coupled to the amino group to form an amide bond in a single step using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (10 mM) as coupling reagent in the presence of N-hydroxysuccinimide (1 mM). After carrying out a competitive immune reaction, antibodies bound to immobilized steroids were estimated by means of a second antibody-enzyme conjugate. The non-specific background was reduced with blocking agents which did not interfere with the immune reaction between antibodies and the steroids coupled to the plastic surface. The following two procedures were effective for this purpose: pretreatment of wells with 0.01% Tween 20 solution followed by 0.5% bovine serum albumin in phosphate buffered saline, and addition of 0.01% Tween 20 to the assay buffer. With this method, the preparation of steroid-enzyme conjugates is unnecessary and optimization of conditions for ELISA procedures can be achieved in a simple manner.

Analysis of urinary protein by immunoblot method using unconcentrated urine in preeclampsia.

In order to investigate the renal change in preeclampsia, molecular weight and specific protein analyses in unconcentrated urine were performed by the immunoblot method. Urine samples were taken from 34 preeclamptic cases (pure type), including 20 severe cases. Polypeptide profiles of urine consisted of four patterns: low MW (L) pattern (tubular damage), high MW (H) pattern (glomerular damage), high and low MW (HL) pattern, and middle MW (M) pattern. The incidences of the HL, H, L, and M patterns were 26.5%, 14.7%, 11.8%, and 47.1%, respectively. The HL pattern was found more frequently in severe proteinuria than in mild proteinuria. High incidences of the HL and H patterns were found in the hypertensive group. Larger amounts of IgM, fibronectin, IgG, and beta 2 microglobulin in urine were confirmed using specific antibodies. Our results suggest that the immunoblot method makes it possible to differentiate glomerular and tubular damages and to evaluate the severity of renal damage in preeclampsia using unconcentrated urine.

Calcitonin-immunoreactive cells are present in the brains of some cyclostomes.

In the brains of four species of cyclostomes (two species each of lampreys and hagfishes), immunoreactive calcitonin-producing cells (iCT cells) were located immunohistochemically by the peroxidase-antiperoxidase method using anti-salmon calcitonin antiserum. In the case of both the adults and the ammocoetes of the brook lamprey (Lampetra reissneri) which lives in freshwater throughout its life, iCT cells were found in two distinct areas: in the pars ventralis hypothalami of the diencephalon and in the torus semicircularis of the mesencephalon. The iCT cells in the diencephalon are classified as bipolar nerve cells, and those in the mesencephalon are classified as multipolar nerve cells. In both the anadromous and catadromous arctic lamprey (Lampetra japonica), iCT cells were present only in the diencephalon, and those were bipolar nerve cells. There seemed to be no differences in the numbers and the immunostainability of the iCT cells, despite the different environments inhabited by the lampreys. In the hagfishes (Eptatretus burgeir and Paramyxine atami) that inhabit seawater throughout their lives, iCT cells were also found only in the diencephalon, although they were very few in number and exhibited poor immunostainability.

Cells showing immunoreactivity for calcitonin or calcitonin gene-related peptide (CGRP) in the central nervous system of some invertebrates.

In the central nervous system of some species of several invertebrate phyla, including land planarians (Platyhelminthes), ribbon worms (Nemertina), slugs (Mollusca), polychaetes, earthworms and leeches (Annelida), pill bugs (Arthropoda), and beard worms (Pogonophora), salmon calcitonin-immunoreactive cells and rat calcitonin gene-related peptide (CGRP)-immunoreactive cells were found by immunohistochemistry. These immunoreactive cells were located in the region surrounding the neuropile, although the sizes of the cells varied according to species. Some of them were round or polygonal and regarded as apolar nerve cells because of their lack of cytoplasmic processes, whereas others were spindle-shaped or elongated, being comparable with unipolar nerve cells because of extension of their cytoplasmic processes in the direction of the neuropile. In some cases, it was noted that the cytoplasmic processes had complicated branches or formed loop-like structures at their ends. These observations suggest that a calcitonin-like or CGRP-like substance is extensively present in invertebrates as well as vertebrates.

Ontogeny of the response of the hypothalamo-pituitary-adrenal axis to maternal immobilization stress in rats.

In this study we investigated the response of the rat fetal hypothalamo-pituitary-adrenal (HPA) axis to an acute maternal stress in late gestation. On day 20 of gestation, pregnant rats were exposed to forced immobilization stress for up to 60 min. In mothers, a significant increase in plasma ACTH and corticosterone(B) was observed at 20 and 60 min. The ACTH content in the maternal pituitary decreased significantly at 60 min. Fetal blood pH was decreased by the maternal stress, showing a hypoxic condition of the fetus. Fetal plasma ACTH increased transiently at 20 min. Fetal plasma B increased at 20 and 60 min. ACTH in the fetal pituitary and the placenta did not show marked changes due to the maternal stress. Pregnant rats on day 18-21 of gestation were subjected to a 20 min maternal stress. In the basal condition without stress, fetal plasma ACTH and B showed parallel ontogenic patterns, having a peak value on day 19 of gestation. Fetal plasma ACTH as well as plasma B were increased significantly by the maternal stress at all points evaluated. These results indicate that fetal hypoxia is important in stress transmission to the fetal HPA axis in this type of maternal stress, and the fetal HPA axis responds to the stress as early as day 18 of gestation.

The effect of epidermal growth factor (EGF) on progestin secretion and cyclic AMP synthesis in cultured luteal cells from pregnant rats.

It is reported that steroid synthesis in ovarian cells is affected by epidermal growth factor (EGF). We cultured luteal cells from pregnant rats for 2 days with or without EGF, followed by incubation of the cells with or without stimulants (hCG, forskolin and dibutyryl cyclic AMP) for 5 hours. The levels of progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) and cyclic AMP (cAMP) in the media were assayed. EGF had no effect on the basal levels of progesterone, 20 alpha-dihydroprogesterone and cAMP, but it suppressed these levels which were increased by the stimulants. We investigated binding capacity of [125I]-EGF to ovarian tissue of pregnant rats. Ovarian tissue had specific binding sites for EGF. The maximum number of binding sites was 2.38 fmol/mg tissue and the Kd value was 0.547 nM. It was indicated that EGF modified the reactivity of luteal cells to stimulants; counteracting the tropic effect of gonadotropins. It was shown that this effect of EGF might be exerted through its receptor in luteal cells.

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats.

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.

Sex-specific effects on the fetal neuroendocrine system during acute stress in late pregnancy of rat and the influence of a simultaneous treatment by tyrosine.

It is well-known that prenatal chronic intermittent stress affects the reproductive system of both sexes. Investigating the effects of an acute maternal stress on the fetal neuroendocrine system, parameters such as hypothalamic catecholamines. CRF, GRF, LH-RH, beta-endorphin, hypophysial beta-endorphin and beta-LPH as well as plasma LH, corticosterone and androstenedione were measured. Pregnant rats of Wistar strain were exposed to restraint stress at day 22 of gestation or to forced immobilization at day 20 of gestation, respectively, and were sacrificed before stress and 10, 30, 60, and 120 min after starting stress. A decrease of fetal hypothalamic catecholamines and an increase of LH-RH content of the hypothalamus as well as of plasma catecholamines were observed under stress on day 22 of gestation. On day 20 of gestation hypothalamic beta-endorphin was depleted in male and unchanged in female fetuses under stress. A depletion of hypothalamic CRF was observed in male fetuses, whereas female fetuses showed an increase of hypothalamic CRF. An increase of GRF was found in fetuses of both sexes. Pituitary opioid content increased in fetuses of both sexes initially, but was depleted secondarily in male fetuses. The LH plasma level was markedly reduced in male, the corticosterone level was elevated in fetuses of both sexes as well as the androstenedione level in female fetuses. A simultaneous treatment of mother animals with tyrosine--a catecholamine precursor--prevented the depletion of hypothalamic and pituitary beta-endorphin as well as in part the reduction of plasma LH levels in male fetuses. Hypothalamic GRF content does not increase under tyrosine treatment in male fetuses, whereas in female fetuses the stress-induced increase of GRF content was rather pronounced under tyrosine than attenuated. These results indicate that fetal hypothalamic neurotransmitters and neurohormones (such as LH-RH, CRF, GRF and opioids) are involved in changing circulating hypophysial and adrenal hormones in fetuses exposed to maternal stress in late pregnancy, whereby sex-specific different pathways might be effective in fetal stress processing. The prenatal administration of tyrosine prevented at least in part--those neurohormonal changes which are affecting the sex-specific brain differentiation.

Inhibition of rat ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase and 17,20 lyase by progestins and danazol.

The site of action of synthetic progestins or danazol in the treatment of endometriosis is considered to be mainly the hypothalamo-pituitary level, but the direct action to the uterine endometrium and the ovary is also suggested. We investigated the effect of these synthetic steroids to rat ovarian steroidogenic enzymes. The effect of norethisterone, levonorgestrel, danazol, gestrinone, desogestrel and 3-keto-desogestrel was studied in vitro. The sources of the enzymes were prepared from ovaries of immature rats treated either with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) for 3 beta-hydroxy steroid dehydrogenase (3 beta-HSD), or with PMS for 17 alpha-hydroxylase and 17,20 lyase. The substrates used were pregnenolone (P5) for 3 beta-HSD, progesterone (P4) for 17 alpha-hydroxylase, and 17 alpha-hydroxy-progesterone (17 alpha-OH-P4) for 17,20 lyase. The substrates were incubated with the enzyme sources and coenzymes, and the products formed were measured. All the steroids inhibited 3 beta-HSD, and the inhibition by gestrinone (Ki = 3.0 microM) and 3-keto-desogestrel (17.5 microM) was particularly marked. Only desogestrel (Ki = 30.3 microM) and danazol (168 microM) inhibited 17 alpha-hydroxylase. All the steroids inhibited 17,20 lyase, and the inhibition by desogestrel (Ki = 0.70 microM), danazol (0.80 microM), and gestrinone (30 microM) was particularly marked.

Radioimmunoassay of 2-hydroxyesterone using antisera raised against antigenic complexes obtained by convenient methods.

Antigenic complexes of 2-hydroxyestrone (2-OHE1) were obtained by Mannich reaction of 2-OHE1 and bovine serum albumin (BSA) and by coupling of 2-OHE1 1-glutathione thioether to BSA using glutaraldehyde. Antiserum raised against the antigen obtained by the Mannich reaction had high affinity (Kd = 3.8 x 10(9) M-1) and relatively high specificity; cross reactivities for estrone, 4-hydroxyestrone and 2-methoxyestrone were 2.1%, 10% and 1.5%, respectively. The other antiserum also had high affinity (4.5 x 10(9) M-1) but its cross reactivities for the above three steroids were more than 100%. Concentrations of 2-hydroxyestrone in human plasma were determined by radioimmunoassay with the more specific antiserum and Sephadex LH-20 chromatography to be less than a minimum detectable amount (less than 10 pg/ml) (men), 20.9 pg/ml (women, proliferation) and 26.0 pg/ml (women, periovulation).