Impact of bridge heterology on functional parameters of ELISA for 17α-methyltestosterone.

In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.

Direct impact of gonadotropins on glucose uptake and storage in preovulatory granulosa cells: Implications in the pathogenesis of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is often associated with higher levels of LH, and arrested ovarian follicular growth. The direct impact of high LH on FSH mediated metabolic responses in PCOS patients is not clearly understood. METHOD: In order to investigate the impact of FSH and LH on glucose metabolism in preovulatory granulosa cells (GCs), we used [U14C]-2 deoxyglucose, D-[U14C]-glucose or 2-NBD glucose to analyse glucose uptake and its incorporation into glycogen. To reproduce the high androgenic potential in PCOS patients, we administered hCG both in vitro and in vivo. The role of IRS-2/PI3K/Akt2 pathway was studied after knockdown with specific siRNA. Immunoprecipitation and specific assays were used for the assessment of IRS-2, glycogen synthase and protein phosphatase 1. Furthermore, we examined the in vivo effects of hCG on FSH mediated glycogen increase in normal and PCOS rat model. HEK293 cells co-expressing FSHR and LHR were used to demonstrate glucose uptake and BRET change by FSH and hCG. RESULTS: In normal human and rat granulosa cells, FSH is more potent than hCG in stimulating glucose uptake, however glycogen synthesis was significantly upregulated only by FSH through increase in activity of glycogen synthase via IRS-2/PI3K/Akt2 pathway. On the contrary, an impaired FSH-stimulated glucose uptake and glycogen synthesis in granulosa cells of PCOS-patients indicated a selective defect in FSHR activation. Further, in normal human granulosa cells, and in immature rat model, the impact of hCG on FSH responses was such that it inhibited the FSH-mediated glucose uptake as well as glycogen synthesis through inhibition of FSH-stimulated IRS-2 expression. These findings were further validated in HEK293 cells overexpressing Flag-LHR and HA-FSHR, where high hCG inhibited the FSH-stimulated glucose uptake. Notably, an increased BRET change was observed in HEK293 cells expressing FSHR-Rluc8 and LHR-Venus possibly suggesting increased heteromerization of LHR and FSHR in the presence of both hCG and FSH in comparison to FSH or hCG alone. CONCLUSION: Our findings confirm a selective attenuation of metabolic responses to FSH such as glucose uptake and glycogen synthesis by high activation level of LHR leading to the inhibition of IRS-2 pathway, resulting in depleted glycogen stores and follicular growth arrest in PCOS patients.

pH-Sensitive Biocompatible Nanoparticles of Paclitaxel-Conjugated Poly(styrene-co-maleic acid) for Anticancer Drug Delivery in Solid Tumors of Syngeneic Mice.

In the present study, we have synthesized poly(styrene-co-maleic anhydride), a biocompatible copolymer that was further conjugated with paclitaxel (PTX) via ester linkage and self-assembled to form poly(styrene-co-maleic acid)-paclitaxel (PSMAC-PTX) nanoparticles (NPs). The in vitro release of PTX from PSMAC-PTX NPs showed a higher release at lower pH than at the physiological pH of 7.4, confirming its pH-dependent release. The cell viability of PSMAC-PTX nanoparticles was evaluated using MTT assay. IC50 values of 9.05-18.43 ng/mL of PTX equivalent were observed in various cancer cell lines after 72 h of incubation. Confocal microscopy, Western blotting, and Flow cytometry results further supported that the cellular uptake and apoptosis of cancer cells with PSMAC-PTX NPs. Pharmacokinetic studies revealed that the conjugation of PTX to the PSMAC co-polymer not only increased the plasma and tumor C(max) of PTX but also prolonged its plasma half-life and retention in tumor via enhanced permeability and retention (EPR) effect. Administration of PSMAC-PTX NPs showed significant tumor growth inhibition with improved apoptosis effects in vivo on Ehrlich Ascites Tumor (EAT)-bearing BALB/c syngeneic mice in comparison with Taxol, without showing any cytotoxicity. On the basis of preliminary results, no subacute toxicity was observed in major organs, tissues and hematological system up to a dosage of 60 mg/kg body weight in mice. Therefore, PSMAC-PTX NPs may be considered as an alternative nanodrug delivery system for the delivery of PTX in solid tumors.

FSH stimulates IRS-2 expression in human granulosa cells through cAMP/SP1, an inoperative FSH action in PCOS patients.

Follicle stimulating hormone (FSH) plays a central role in growth and differentiation of ovarian follicles. A plethora of information exists on molecular aspects of FSH responses but little is known about the mechanisms involved in its cross-talk with insulin/IGF-1 pathways implicated in the coordination of energy homeostasis in preovulatory granulosa cells (GCs). In this study, we hypothesized that FSH may regulate IRS-2 expression and thereby maintain the energy balance in GCs. We demonstrate here that FSH specifically increases IRS-2 expression in human and rat GCs. FSH-stimulated IRS-2 expression was inhibited by actinomycin D or cycloheximide. Furthermore, FSH decreases IRS-2 mRNA degradation indicating post-transcriptional stabilization. Herein, we demonstrate a role of cAMP pathway in the activation of IRS-2 expression by FSH. Scan and activity analysis of IRS-2 promoter demonstrated that FSH regulates IRS-2 expression through SP1 binding sites. FSH stimulates SP1 translocation into nucleus and its binding to IRS-2 promoter. These results are corroborated by the fact that siRNA mediated knockdown of IRS-2 decreased the FSH-stimulated PI3K activity, p-Akt levels, GLUT4 translocation and glucose uptake. However, FSH was not able to increase IRS-2 expression in GCs from PCOS women undergoing IVF. Interestingly, IRS-2 mRNA expression was downregulated in GCs from the PCOS rat model. Taken together, our findings establish that FSH induces IRS-2 expression and thereby activates PI3K, Akt and glucose uptake. Crucially, our data confirms a molecular defect in FSH action in PCOS GCs which may cause deceleration of metabolism and follicular growth leading to infertility. These results lend support for a therapeutic potential of IRS-2 in the management of PCOS.

Selective capturing and detection of Salmonella typhi on polycarbonate membrane using bioconjugated quantum dots.

Present work demonstrates the utilization of surface modified polycarbonate (PC) membrane as solid phase and antibody conjugated CdSe/ZnS quantum dots (QDs) as fluorescent label for the sensitive and selective detection of Salmonella typhi (S. typhi) in water in a period of 2.5h. PC membrane was surface modified with glycine and activated by EDC/NHS for immobilization of S. typhi specific IgG. Antibody immobilized porous PC membrane was incubated with bacteria contaminated water for immunocapturing of S. typhi. Antibody conjugated QDs were also prepared by using carbodiimide chemistry. Both modified PC membrane and quantum dots were characterized by using various modern analytical tools. It was estimated that 1.95 molecules of QDs were successfully bio-conjugated per unit of IgG. PC membrane with captured bacteria was incubated with prepared IgG conjugated QDs for the formation of sandwich complex. Analysis of the regions of interest (ROI) in fluorescent micrographs showed that newly developed method based on PC and fluorescent QDs has 100 times higher detection sensitivity (100 cells/mL) as compared with detection using conventional dye (FITC) based methods.

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs.

Non-specific L-type calcium channel blockers, such as verapamil (> or =50 microM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca(2+)-deficient medium. However, the effects of extracellular Ca(2+) on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca(2+)-deficient medium. The initiation of apoptotic features was observed at 50 microM of verapamil. Extracellular Ca(2+) (1.80 mM) reduced intracellular H2O2 level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 microM, but not higher concentrations of > or =100 microM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca(2+) ions have a role during SEA and protect against verapamil induced apoptosis in aged rat eggs.

Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis.

The objective was to find out the functional roles of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H(2)O(2), total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H(2)O(2) induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H(2)O(2), reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H(2)O(2) and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide.

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

Development of colorimetric enzyme-linked immunosorbent assay for human chorionic gonadotropin.

The present study demonstrated the development of a solid phase competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of human chorionic gonadotropin (hCG) in serum and urine. Polyclonal antisera raised against the beta- subunit of peak-I hCG was used in the assay. The Peak-IA hCG-penicillinase was used as tracer. The performance of this antiserum and tracer was compared against hCG-beta antisera of NIH, USA and penicillinase conjugated to hCG-beta obtained from NIH, respectively. Almost parallel standard curves were obtained in both cases, suggesting that these antisera and enzyme label have much potential for developing ELISA system. To the anti-rabbit gamma globulin (ARGG) coated polystyrene tubes, standard or serum or urine samples (50 microL), 100 microL of hCG-beta antiserum, 100 microL of peak-I(A) hCG-penicillinase conjugate and 350 microL of assay buffer were incubated at 37 degrees C for 2 hours. Bound enzyme activity was measured using Penicilline V as substrate. In this new strategy, locally available polystyrene tubes were ground from inside and coated with ARGG. The sensitivity of the assay was 17 mIU/mL in urine and 18 mIU/mL in serum. The intra-assay and inter-assay coefficients of variation (CVs) appeared to be within acceptable limits of 10%. The serum and urinary hCG values, obtained by this method, correlated well with those obtained by radioimmunoassay (RIA) r = 0.98 (n = 100 for serum samples; n = 250 for urinary samples).

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro.

Hydrogen peroxide (H2O2) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H2O2 with or without caspase-3 inhibitor for various times. The treatment of H2O2 induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H2O2 treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H2O2 treatment. The shrinkage was induced by H2O2 in a dose- and time-dependent manner in both immature and mature oocytes. Although, H2O2-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H2O2 after 1.0 hr of culture. Treatment of H2O2 induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H2O2 induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.

Isolation of hCG and its characterization by radioimmunoassay, enzyme-immunoassay, and radio-receptor assay.

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45-75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G-100 chromatography followed by ion-exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G-200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of hCG-alpha (mol. wt. 19,539D) and hCG-beta (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera.

Development of polyclonal antisera is still a choice in some hard-pressed budget laboratories. In the present study, an attempt was made to isolate alpha- and beta-subunits from peak-I hCG, generation of polyclonal antisera and their characterization. The anti-hCG-a antisera showed titres of 1: 8000 and anti-hCG-beta antisera 1: 16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG-beta antiserum demonstrated no cross-reaction with several hormones tested in the present study, except for hCG-beta and hCG, thus eliciting a highly specific hCG-beta antiserum.

Spermicidal activity of Azadirachta indica (neem) leaf extract.

The present study was carried out to evaluate the effective concentration of aqueous extract of old and tender Azadirachta indica (neem) leaves to immobilize and kill 100% human spermatozoa within 20 s. Sander-Cramer test was used to study the spermicidal activity of neem leaf extract. Under the test conditions, minimum effective spermicidal concentrations for tender and old leaf extracts were 2.91 +/- 0.669 mg/million sperm and 2.75 +/- 0.754 mg/million sperm, respectively. The effect of extracts on morphology and viability of sperm was also studied and no change was observed in morphology of head, mid-piece and tail and no viable sperm seen. The leaf extracts were found to be water soluble and carbohydrate in nature. The effect of different concentrations of extracts (old and tender) on percentage motility of the sperm was also studied. With an increase in concentration, there is a linear decrease in percentage motility, becoming zero at a 3-mg dose within 20 s.

One step enzyme linked immunosorbent assay for direct estimation of serum cortisol.

One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol in human serum is described. Cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) was used as an immunogen and cortisol-21-hemisuccinate-horse radish peroxidase (cortisol-21-HS-HRP) was used as a tracer. To the cortisol antibody coated microtiter wells, standards or serum samples (25 microl) along with cortisol-HRP conjugate (100 microl) were incubated for 2 hours at 37 degrees C. Bound enzyme activity was measured by, using TMB/H2O2 as a substrate. In this new strategy, chilled acetone stripped pooled human serum and sodium salicylate were used for preparing the standards and blocking the cortisol binding globulin (CBG), respectively. The sensitivity of the assay was .28 microg/100ml. The intraassay and interassay coefficient of variations (CVs) were ranged from 1.3% to 9.3% and 6.8% to 12.3 %, respectively. The analytical recoveries were 94% to 101.5%. The serum cortisol values, obtained by this method were correlated well with those, obtained by radioimmunoassay; r=0.95 (n=52).

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.

Enzyme immunosorbant assay of oestradiol in unextracted plasma using penicillinase as label.

An enzyme-linked immunosorbant assay (ELISA) for measuring oestradiol directly in plasma without extraction utilizing antibodies raised against oestradiol-3-(O-carboxymethyl) ether-bovine serum albumin conjugate, and oestradiol-6-(O-carboxymethyl) oxime linked to penicillinase (EC 3.5.2.6) as a marker was developed. Polyvinyl 96-well microtitre plates were used for immobilization of anti-oestradiol IgG. Standards of oestradiol (92 to 9,190 pmol/l were prepared in oestradiol-free plasma and 8-anilino-1-naphthalene sulphonic acid (8-ANS, 5 mg/ml of 10 mmol/l PBS) was added to the microtitre plate wells to displace oestradiol from plasma binding proteins. The assay had a lower limit of detection of 92 pmol/l plasma and could be performed within 4 h. Comparison of oestradiol values of 51 plasma specimens obtained by ELISA with those of radioimmunoassay (RIA), in which oestradiol was extracted with diethyl ether, showed good correlation (y = 0.786x + 0.03; r = 0.900).

Enzyme immunosorbent assay of prolactin with penicillinase as label.

This competitive, rapid, and sensitive enzyme immunosorbent assay for measuring human prolactin in plasma involves penicillinase (EC 3.5.2.6) as label. Microtiter plate wells coated with goat anti-rabbit gamma globulin are filled with antibody to prolactin and plasma sample or reference prolactin and incubated with penicillinase-labeled prolactin for 1 h at 37 degrees C. The enzyme activity of the bound complex is then measured. The assay is as sensitive as radioimmunoassay, the detection limit being 2.5 ng of prolactin per milliliter of plasma. The plasma prolactin values obtained by enzyme immunoassay correlated well with those determined by radioimmunoassay: r = 0.98, slope = 1.00, intercept = 1.11 ng/mL (n = 53).

Hormonal control of implantation in guinea pigs.

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).

Enzyme immunoassay of cortisol in human plasma using penicillinase as label.

An enzyme immunoassay for cortisol in human plasma using an antiserum raised against cortisol-3-O-carboxy-methyloxime bovine serum albumin and cortisol-21-hemisuccinate conjugated to penicillinase as tracer is described. Although employing immunoassay plates for separation of antigen-antibody complex from the free components was less time consuming, the slope and sensitivity of the standard curve were improved by the addition of goat anti-rabbit gamma globulin for precipitating the complex. There was good correlation between radioimmunoassay and enzyme immunoassay results obtained for cortisol levels present in normal human plasma.