Radioimmunoassay of ovine and bovine serum progesterone without extraction and chromatography

A procedure for the radioimmunoassay of ovine and bovine serum progesterone is described which does not require extraction and chromatography. Serum samples are assayed directly, and a highly specific antiserum that was prepared in rabbits against 11 alpha-hydroxyprogesterone conjugated to bovine serum albumin is used. Interference from serum binding proteins is alleviated by use of a phosphate buffer containing 5% BSA and separation of bound and free tritiated progesterone by a double antibody procedure. Serum samples are assayed in a mini-vial, the bound fraction (double antibody precipitate) is mixed with scintillation solution and the radioactivity is counted in the same vial. The assay procedure is sensitive (10 pg, 100 pg/ml) and has acceptable accuracy and precision. Because there is no extraction or chromatography, serum progesterone is not lost. Most important, the procedure is specific for progesterone and measures serum progesterone concentrations in the ewe and cow which are comparable with those obtained with conventional assay techniques. The progesterone assay described herein provides a rapid, economical procedure that can facilitate the study of ovarian cyclicity and aid in the early diagnosis of pregnancy.     

Tumouricidal activity of gilthead seabream (Sparus aurata L.) natural cytotoxic cells: the role played in vitro and in vivo by retinol acetate

The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was evaluated after in vitro incubation with retinol acetate as vitamin A source, and in samples taken from specimens receiving an intraperitoneal injection or a diet supplemented with this vitamin. Isolated leucocytes were incubated with 0 to 10(-10)m all-trans-retinol acetate-supplemented culture medium for 0, 6 or 24h and assayed for their tumouricidal activity which was found to increase for all the assayed concentrations and incubation times. Seabream specimens were intraperitoneally injected with 0 (control), 1.75 or 5.25 micro g retinol acetate 100 g(-1) biomass and sampled 1, 3 or 5 days post-injection. Leucocyte natural cytotoxic activity increased in a dose-dependent manner 1 and 3 days post-injection. When fish were fed a commercial diet supplemented with 0 (control), 50, 150 or 300 mg retinol acetate kg(-1) diet for 1, 2, 4 or 6 weeks, only fish which had been fed the highest supplement for 2 weeks showed any increase in head-kidney leucocyte cytotoxic activity. Serum was isolated and analysed for all-trans-retinol concentration by reverse-phase high-pressure-liquid-chromatography. The normal level was about 0.4 micro g ml(-1) serum, while treatment for 1 to 4 weeks with vitamin A increased this level.In conclusion, retinol acetate increases gilthead seabream head-kidney leucocyte cytotoxic activity both in vitro and in vivo.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Assessing vitamin D status of callitrichids: Baseline data from wild cotton-top tamarins (Saguinus oedipus) in Colombia

Bone disease related to vitamin D deficiency is an insidious problem in captive colonies of callitrichids. Currently a diagnosis of vitamin D deficiency may be made after the appearance of clinical pathology, usually bone deformities or fractures. The development of assays for vitamin D metabolites suggests it may be possible to detect incipient vitamin D deficiency before animals are adversely affected. However, there are few data on normative levels of these metabolites in any nonhuman primates. We collected blood samples from 18 wild cotton-top tamarins (Saguinus oedipus) from Colombia and assayed them for 25-hydroxy vitamin D(25-OH-D), the major circulating form of the vitamin, which is believed to be a good indicator of status. Serum concentrations of 25-OH-D averaged 76.4 ng/ml (range 25.5–120 ng/ml). This is high compared with human norms (10–55 ng/ml), but the range is lower than that reported in the literature for captive callitrichids. Juveniles had higher serum concentrations than did adults, and pregnant females had lower concentrations than did nonpregnant females. These data confirm that healthy callitrichids have higher circulating levels of 25-OH-D than do humans, but they suggest that the extremely high levels found in some captive animals (300–600 ng/ml) may be above normal. We propose that serum concentrations of 25-OH-D of 50–120 ng/ml can be considered normal for cotton-top tamarins and perhaps other callitrichids. If serum values much below 50 ng/ml are found during clinical evaluation, the possibility of incipient vitamin D deficiency should be considered. Zoo Biol 16:39–46, 1997. © 1997 Wiley-Liss, Inc.     

Quantification of apolipoproteins in rat serum and in cultured rat hepatocytes by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure apolipoproteins in rat serum. Nondelipidated whole serum was heat-treated at 52 degrees C for 3 h in phosphate-buffered saline containing 0.1% Tween-20 before assay. Monospecific rabbit anti-rat apolipoprotein antibodies were added to 96-well polystyrene microtiter plates which had been coated with purified rat serum apolipoproteins or unknown samples. After incubation and washing, goat anti-rabbit serum antibodies conjugated with horseradish peroxidase were added to the plates and incubated. The bound peroxidase activity was assayed after further washing. Serum apolipoprotein concentrations were calculated by comparison against purified standards that were assayed simultaneously with the unknown samples. The intraassay coefficients of variation for apolipoprotein AI, E, and AIV (Apo AI, E, and AIV) were 2.3, 4.4, and 5.3%, and interassay coefficients of variation were 6.1, 5.5, and 7.9%, respectively. The ELISA assay is sensitive to nanogram quantities of rat serum apolipoproteins and the results agree well with those measured by densitometry. The serum concentrations of Apo AI, E, and AIV of a normal fed rat were found to be 504 +/- 8, 413 +/- 20, and 262 +/- 20 micrograms/ml, respectively. When cultured as monolayers in Waymouth's medium for 1 day, rat hepatocytes secreted Apo AI, E, and AIV at rates of 2.51, 61.8, and 48.9 ng protein/mg cell protein/h.     

Electroimmunoassay of rat apolipoproteins A-I, A-IV, and E. A procedure for sample treatment to increase the sensitivity in diluted fractions

Methods for the quantitative determination of rat apolipoproteins A-I, A-IV, and E by electroimmunoassay are described. Apolipoproteins present in diluted samples of biological fluids (approx. 2 ml) were concentrated by precipitation with deoxycholate and trichloroacetic acid. The protein pellets were solubilized in 0.1 ml of 0.5 M NaOH and these samples were delipidated with tetramethylurea and assayed. This protocol enables the measurement of apolipoprotein concentrations that are at least 10 times lower than normally detectable; 0.2 micrograms of apolipoprotein A-IV, 0.2 micrograms of apolipoprotein A-I, and 0.8 micrograms of apolipoprotein E can be easily detected in samples of 2 ml.     

Transplacental transport of alpha 2-macroglobulin (alpha 2M) and induction of alpha 2M in maternal and neonatal rats with acute inflammation.

The aims of this study were to investigate transplacental transport of alpha 2-macroglobulin (alpha 2M) in rats in rats and to examine the degree of alpha 2M induction in maternal and neonatal rats with acute inflammation. Serum was collected from healthy pregnant CD (IGS) rats, neonates of the pregnant rats and their cord blood. Additional serum samples were obtained from pregnant rats inoculated with an inflammatory agent, turpentine oil, their neonates and cord blood, and neonates inoculated with turpentine oil. The serum levels of alpha 2M were measured by means of an enzyme-linked immunosorbent assay. The average serum levels of alpha 2M in healthy neonates and cord blood were about 380 micrograms/ml. Serum a2M level in neonates inoculated with turpentine oil averaged about 580 micrograms/ml. Serum alpha 2M levels in maternal rats inoculated with turpentine oil, neonates from those rats and their cord blood were elevated, the values being 2,000 micrograms/ml or higher. It was demonstrated that induction of alpha 2M in neonatal rats was lower than in maternal rats when inoculated with turpentine oil. These results suggest that alpha 2M is transplacentally transported from maternal rats to fetal ones.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

Effects of steroid hormones in fetal bovine serum on plating ang cloning of human cells in vitro.

Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-beta-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 mug per ml of progesterone. The addition to non-supporative sera of 0.1 mug per ml of 17-beta-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro.     

Effect of zearalenone and estradiol benzoate on serum concentrations of LH, FSH and prolactin in ovariectomized gilts

Six ovariectomized gilts were given zearalenone (Z), estradiol benzoate (EB) or vehicle in a replicated 3 x 3 Latin square design. Zearalenone was added to 2.3 kg of a corn-soybean ration at a dose of 1 mg Z/kg body weight; EB was given intramuscularly at 0.1 mg EB/kg body weight. Control gilts received vehicle solvent for both Z and EB. Blood samples were collected from indwelling jugular cannulas at 6-h intervals for 48 h before Z, EB or vehicle was given. After treatment, blood samples were drawn at 6-h intervals for an additional 84 h. Serum concentrations of luteinizing hormone (LH) decreased (P<0.001) from 4.67 ng/ml to 0.29 ng/ml within 6 h of EB. From 54 to 84 h after EB, serum concentrations of LH rose to 15.60 ng/ml (P<0.001). Serum concentrations of LH were reduced (P<0.001) in a similar pattern after Z (3.70 ng/ml to 0.49 ng/ml), but a rise in serum LH was not observed 54 to 84 h after Z (1.30 ng/ml). Serum concentrations of LH remained unchanged (P=0.55) in gilts given vehicle. Serum concentrations of follicle stimulating hormone (FSH) were suppressed (P<0.03) at 6 h in EB (19.10 vs 11.35 ng/ml) and Z gilts (16.16 vs 11.41 ng/ml) but remained unchanged in vehicle gilts. Serum concentrations of FSH did not change in EB or Z gilts during the next 36 h. These data indicate that the suppressive action of Z on serum concentrations of LH and FSH was similar to that of EB, while the biphasic stimulatory effect of EB for LH was not manifested by Z.     

Human alpha-L-fucosidase: a common polymorphic variant for low serum enzyme activity, studies of serum and leukocyte enzyme.

Serum alpha-L-fucosidase was assayed in 422 individuals. Extremely low levels of enzyme activity were found in 26 individuals. This variant enzyme has increased heat lability. The remaining samples exhibited a bimodal frequency distribution. Family studies suggest that the variant is inherited as a Mendelian recessive character. The variant leukocyte enzyme exhibits normal activity yet increased heat lability. Serum and leukocyte samples from individuals who do not possess the low activity variant are heterogeneous with respect to the heat lability of alpha-L-fucosidase. This common polymorphism may be useful for mapping purposes in family studies.     

Hyperprolactinemia and sexual function in men

Male hyperprolactinemia (HPRL) is known to induce different types of sexual dysfunctions. In order to determine the incidence of HPRL among patients referred for sexual dysfunction, serum prolactin (PRL) was assayed in 1053 clinically idiopathic cases. Among 850 cases complaining of erectile impotence, 10 with marked HPRL (1.1%, PRL above 35 ng/ml) were found, of whom 6 cases were associated with a pituitary adenoma. 17 mild HPRL (2%, PRL 20-35 ng/ml) were also found. Among 124 cases with premature ejaculation, 13 (10%) mild HPRL were found. Serum PRL was normal in 51 cases complaining of an ejaculation without orgasm, and 27 patients exclusively complaining of reduced sexual desire. Our results lay stress on the fact that serum PRL must be assayed in every case of clinically idiopathic erectile impotence. Indeed, 5 of the 10 marked HPRL patients would have been misdiagnosed if we had only assayed this hormone when plasma testosterone was below the normal range. Moreover, in order to shed some light on the mechanisms by which HPRL disturbs male sexual function, the sexual behaviour of 17 markedly HPRL males was compared to their serum levels of PRL and testosterone, first before treatment, then at regular intervals during treatment. Our main conclusion is that impotence cannot be totally explained by a decrease in plasma testosterone, because this steroid hormone was within the normal range 7 of the 16 impotent patients. Moreover, when serum PRL was lowered by bromocriptine, 6 patients recovered their potency before plasma testosterone clearly increased, and in 3 of those patients before it reached the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

A sensitive enzyme immunoassay for human basic fibroblast growth factor

A sensitive sandwich enzyme immunoassay for human basic fibroblast growth factor (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic fibroblast growth factor, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in 16 out of 57 samples at range 30 approximately 206 pg/ml.     

Sensitive assay for verapamil in plasma using gas-liquid chromatography with nitrogen-phosphorus detection

A sensitive gas-chromatographic method for quantitative analysis of verapamil in human plasma is described. The method involves a single extraction procedure, followed by separation on a capillary column and detection with a nitrogen-phosphorus detector. The detection limit, based upon an assayed plasma volume of 0.5 ml, is 2 ng/ml. The standard curve is linear in the concentration range of 2 to 1000 ng/ml. The recovery of verapamil by pentane-isopropanol extraction was found to be 95%. Zipeprol is used as the internal standard. No interference from drugs needed for the associated cancer therapy has been found. Serum verapamil concentrations are determined by this method in fourteen cancer patients undergoing treatment with adriamycin.     

A comparison of the indirect haemagglutination test with the toxin neutralization test for the estimation of diphtheria antitoxin in mouse sera

Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.     

Sensitive and specific determination of clindamycin in human serum and bone tissue applying liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

A method for the quantification of clindamycin in human serum and in human bone tissue samples applying high-performance liquid chromatography with atmospheric pressure chemical ionization–mass spectrometry (APCI–MS) is presented. Lincomycin is used as the internal standard. Serum samples are prepared only by protein precipitation with acetonitrile. Bone tissue samples have to be crushed and homogenized in extraction buffer prior to analysis. The chromatographic separation is achieved on an RP-18 stationary phase with 0.02% trifluoroacetic acid in water 60%/acetonitrile 40% v/v as mobile phase. The limits of quantification are 0.1 μg/ml for serum samples and 0.1 μg/g for bone tissue samples. The coefficients of variation for the assays are 4.48 and 8.41% at the limit of quantification for serum and bone tissue samples, respectively. Bone tissue samples as small as 50 mg can be used.     

An improved heparin assay which is insensitive to platelet factor 4

A new reagent for the determination of heparin in plasma has been developed. In the assay heparin which was bound to platelet factor 4 is also measured. That is why samples, which have to be assayed for heparin with this reagent, do not need any special pretreatment like fast and cooled processing in order to prevent release of platelet factor 4 from platelets. Heparin can be assayed in samples anticoagulated with citrate which are used routinely for the determination of other coagulation parameters like PT or aPTT. Freezing prior to the assay is possible and does not influence the result. The assay is based on the inactivation of factor Xa by antithrombin III which is catalysed by heparin or smaller fragments of it. It can therefore be used for the determination of heparins of low molecular weight, too. The sample is first mixed with AT III in order to compensate for a potentially decreased level in the probe. Then the F Xa reagent is added, which releases bound heparin from plasma proteins like platelet factor 4 by an added polysulfated dextran simultaneously to the onset of the inhibitory reaction towards F Xa. Free and secondarily released heparin are then available for determination. After a defined period of time a substrate for F Xa is added and the remaining activity is measured in a photometer. An incubation time of 1 min or 3 min is used for the normal range of 0.1 to 1 U/ml or the low dose range from 0.01 to 0.3 U/ml heparin, respectively.     

Serologic survey for transmissible gastroenteritis virus neutralizing antibodies in selected feral and domestic swine sera in the southern United States

Serum samples collected from feral and domestic swine (Sus scrofa) in Florida and feral swine in Georgia and Texas were assayed by plaque reduction for their virus neutralizing (VN) antibodies against the porcine transmissible gastroenteritis virus (TGE). None of 560 samples collected from feral swine contained VN antibodies for TGE virus, but experimentally infected feral swine seroconverted. None of 665 samples from domestic swine contained TGE-VN antibodies. These results indicate feral swine are not a significant reservoir for TGE virus in southern states, but are capable of becoming infected and developing VN antibodies against TGE.     

Oxytocin levels during breast-feeding in established lactation

Serial blood samples were taken from eight lactating women while they were nursing their babies 1–4 months postpartum, and from four lactating controls while they were not nursing. The plasma was assayed for oxytocin by radioimmunoassay after extraction with activated Vycor glass powder. In the suckling mothers mean plasma oxytocin rose from 5.4 pg/ml before nursing to 13.0 pg/ml during nursing. Oxytocin levels changed rapidly from minute to minute, with individual peaks as high as 54 pg/ml. Oxytocin levels in the control mothers averaged 4.4 pg/ml.