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1.
The measurement of hormones in fecal samples allows for the noninvasive assessment of the endocrine status of free-ranging primates. However, procedures and techniques for hormone analysis in feces must be validated, both analytically and physiologically. Few studies have addressed the endocrinology of black howler monkeys (Alouatta pigra). Due to its conservation status, direct handling of individuals from this species and invasive sample collection are highly regulated, and therefore traditional methods for the validation of hormone assays, such as pharmacological challenges, are not allowed. As a consequence, sometimes studies of the fecal hormones of free-ranging black howler monkeys do not report physiological validations and therefore the biological reliability of such measurements cannot be assessed. In order to stimulate future research with this species, the present study aimed at providing methodological bases for fecal endocrine monitoring. Specifically, we compared the validity of two immunoassays (radioimmunoassays, RIA; solid-phase chemiluminescent enzyme immunoassay, SPCEI) performed with commercial kits to measure cortisol, testosterone, estradiol, and progesterone; and demonstrate how the physiological functions of these steroid hormones can be determined through non-pharmacological validations. We found no differences between the analytical validity of RIA and SPCEI assays to measure cortisol and testosterone, whereas for estradiol and progesterone RIA showed better results. Concerning the physiological validation of our assays, we demonstrated that: (1) comparisons between pre- and post-stress situations may be used to assess cortisol response, (2) comparisons between females and males may be used to assess variation in testosterone levels, and (3) comparisons between pregnant and non-pregnant females may be used to determine variation in estradiol and progesterone activity. The analytical and physiological validations that we performed demonstrate that there are currently commercial kits that allow for correct endocrine monitoring of this species, and that there are non-pharmacological alternatives to assess the biological validity of hormone measurements.  相似文献   

2.
Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown not to cross-react with human thyroid stimulating hormone, alpha-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specificity for determining PAP isoenzyme 2 in human serum.  相似文献   

3.
Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.  相似文献   

4.
Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.  相似文献   

5.
The diagnostic value of the first experimental production batches of assay kit "DIAGN-A-HEP", produced at the Institute of Poliomyelitis and Viral Encephalitides (USSR Acad. Med. Sci.) and intended for the determination of IgM to hepatitis A virus (HAV) in the enzyme immunoassay (EIA), has been studied in comparison with that of the internationally known and widely approved commercial EIA system "HAVAB-MEIA" for the determination of antibodies to HAV (the product of Abbott, USA). The study has revealed that the EIA kit "DIAGN-A-HEP" is highly sensitive and specific, and the diagnostic value of this kit is not inferior to that of the commercial assay system "HAVAB-MEIA". On the basis of this study the use of the EIA kits "DIAGN-A-HEP" in medical practice has been allowed by the decree of the Ministry of Health of the USSR.  相似文献   

6.
A combined radioimmunoassay (RIA) for the measurement of the anterior pituitary proteins luteinizing hormone (LH) and prolactin (PRL) is described and compared with individual RIAs for these hormones. The standard curves and the sample values for LH and PRL were identical when determined in a combined or in an individual RIA. This technique may prove useful to a number of laboratories where it is desirable to determine levels of more than one hormone in limited sample volumes.  相似文献   

7.
Commercially available enzyme immunoassay (EIA) kits were validated for measuring steroid hormone concentrations in blood plasma from three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus. A minimum of 5 µl plasma was required to estimate hormone concentrations with both kits. These EIA kits are a simple method requiring minimal equipment, for measuring hormone profiles under field conditions.  相似文献   

8.
We examined the effect of endogenous immunoglobulins (G, A and M) and albumin on the measurement of thyroid hormones by different methods, including a new non-isotopic immunoassay of free thyroxine (FT4) and free triiodothyronine (FT3), in a large number of patients with non-thyroidal illness (NTI). Variations in serum protein concentrations can affect the results of radioimmunoassay of human thyroid hormones and thyroxine binding globulin (TBG). Our data revealed that in patients with non-thyroidal illness, when fluctuations in serum gamma-globulin occurred the T3/TBG and T4/TBG ratios altered. Consequently, when patients are suffering from non-thyroidal illness with changing gamma-globulin levels, clinical scientists should take care when they use T3/TBG and T4/TBG ratios as a substitute for FT3 or FT4 estimation. We found FT4 and FT3 (determined with Amerlex-M kits) T3 and the T3/TBG ratio were altered inversely due to the difference in the serum gamma-globulin levels. A recently developed enhanced luminescence enzyme immunoassay for FT3 and FT4 (Amerlite FT3 and FT4 kits) provides more reliable and accurate results, because of its resistance to interference, especially from albumin and gamma-globulin.  相似文献   

9.
Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.  相似文献   

10.

Background

Accurate and reliable laboratory-based assays are needed for estimating HIV-1 incidence from cross-sectional samples. We recently described the development of a customized, HIV-1-specific Bio-Plex assay that allows for the measurement of HIV-specific antibody levels and avidity to multiple analytes for improved HIV-1 incidence estimates.

Methods

To assess intra- and inter-laboratory assay performance, prototype multiplex kits were developed and evaluated by three distinct laboratories. Longitudinal seroconversion specimens were tested in parallel by each laboratory and kit performance was compared to that of an in-house assay. Additionally, the ability of the kit to distinguish recent from long-term HIV-1 infection, as compared to the in-house assay, was determined by comparing the reactivity of known recent (infected <6 months) and long-term (infected >12 months) drug naïve specimens.

Results

Although the range of reactivity for each analyte varied between the prototype kit and in-house assay, a measurable distinction in reactivity between recent and long-term specimens was observed with both assays in all three laboratories. Additionally, kit performance was consistent between all three laboratories. The intra-assay coefficient of variation (CV), between sample replicates for all laboratories, ranged from 0.5% to 6.1%. The inter-laboratory CVs ranged from 8.5% to 21.3% for gp160-avidity index (a) and gp120-normalized mean fluorescent intensity (MFI) value (n), respectively.

Conclusion

We demonstrate the feasibility of producing a multiplex kit for measuring HIV antibody levels and avidity, with the potential for improved incidence estimates based on multi-analyte algorithms. The availability of a commercial kit will facilitate the transfer of technology among diverse laboratories for widespread assay use.  相似文献   

11.
To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.  相似文献   

12.
Two variants of sandwich-type ELISA (Enzyme Linked Immunosorbent Assay) kits for HBsAg detection (Sevatest ELISA HBsAg Macro I and Sevatest ELISA HBsAg Micro I) in human sera and plasmas were developed. As the solid phase, the ELISA Macro kit and ELISA Micro kit make use of polystyrene microtubes, and polystyrene microtitration plates, respectively, of Czechoslovak production (Koh-i-noor, Dalecín). Capture anti HBs antibody for adsorption to solid phase and rabbit anti HBs antibody for labelling with horse-radish peroxidase were prepared for both tests. The sensitivity of both ELISA kits for HBsAg, equal to approx. 2 ng/ml, was determined by titrating six selected HBsAg-positive sera and the WHO Agk 76 panel of HBsAg-positive sera and the results were compared with those obtained by ELISA, RIA (Radioimmunoassay) and RPHA (Reverse passive hemagglutination) kits of different producers and by counter-immunoelectrophoresis (CIEP). The sensitivity of the new ELISA kits was comparable to that of other producers' ELISA kits, higher than that of RPHA kits and only a little lower than that of RIA kits. A set of sera of patients hospitalised with different diagnoses was tested for HBsAg. The detection rate by ELISA Macro kit 2.8 and 1.5 times higher than by CIEP and RPHA (Raphadex B), respectively, and 1.1 time lower than by RIA (Austria II).  相似文献   

13.
One of the most clearly defined endocrine changes during the parr-smolt transformation of anadromous salmonids is an increase in plasma levels of thyroid hormones. The role of pineal hormone melatonin in timing and synchronisation of smoltification is widely discussed. The effect of administration of exogenous thyroxine (T4) on plasma melatonin was investigated in juvenile Atlantic salmon (Salmo salar) at the early stages of parr-smolt transformation. Fish were kept in fresh water under simulated-natural photoperiod and exposed to exogenous T4. Fish were sampled at 12.00 and 24.00 h from treatment and control tanks, 2 and 14 days after treatment started. Plasma melatonin and L-thyroxine were measured using RIA and competitive enzyme immunoassay, respectively. After 2 days of T4 treatment, marked difference in plasma melatonin concentration measured at 12.00 and 24.00 h was still observed in both groups. However, 2-week exposure to T4 caused a reduction in night-time plasma melatonin level and thus, probably, inhibited melatonin related time-keeping system in juvenile salmon. Additional studies are needed to clarify the mechanism of the described phenomenon.  相似文献   

14.
Various methods have been proposed for the selection of suitable sturgeon broodstock for artificial reproduction. In this study, serum levels of reproductive hormones, namely sturgeon homologues of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2) and progestrone (P), were measured by radioimmunoassay (RIA) in 36 mature broodstock before and after injection of pituitary extract, and the results correlated with sex, age and oocyte polarization index (p). Mature males were significantly younger than females, and showed significant differences in serum P and T levels following pituitary extract injection, although not before that. A significant difference was found between LH, E2 and T before and after injection in the female group. There was also a significant difference between serum levels of T and P in the fertile and infertile groups. Stepwise discriminant function analysis was used to discriminate between fertile and infertile groups. The function had 93,8% overall correct classification, i.e. amongst 100 female broodstock fish, almost 94% of them would be correctly allocated to the relevant group. So the proposed model provides a reliable method for selecting suitable broodstock fish.  相似文献   

15.
Male black tufted-ear marmosets (Callithrix kuhlii) contribute to the rearing of their offspring. Here we evaluated predictions of hypotheses suggesting that (1) T and E2 influence infant-care behavior in male marmosets, (2) levels of T and E2 are modulated by paternal experience, and (3) paternal behavior and levels of T and E2 in male marmosets covary with stress. We observed the behavior of marmosets in their family groups following the birth of infants and evaluated urinary concentrations of T, E2, and the stress hormone cortisol (CORT) among fathers before and after the birth of young. Urinary levels of T, E2, and CORT were lower among males who carried infants at high rates than males who carried at low rates, and T and CORT levels were negatively correlated with carrying rates across all males. Males had significantly lower T levels while carrying the second compared to the first litter and slightly lower rates of infant-carrying, possibly due to assistance provided by offspring of the first litter. There were increases in CORT levels of fathers after the birth of the first litter, but decreases in CORT after the birth of the second. Our results suggest a relationship in C. kuhlii between paternal behavior, hormones, and paternal experience. Rates of infant-carrying appear to be linked to hormone levels, and hormone levels in turn are affected by experience caring for young. Our data also suggest that T, E2, and CORT have synergistic influences on infant-carrying behavior or alternatively that associations between T and E2 and rates of infant-carrying are influenced by stress or other glucocorticoid-related variables. Finally, we propose a hypothesis suggesting that experience-related changes in hormones reinforce the commitment of males to successful breeding partnerships.  相似文献   

16.
Leptin is an adipocyte-derived peptide hormone that acts on the brain and regulates food intake and energy balance. Several previous reports have suggested that overwintering raccoon dogs Nyctereutes procyonoides are able to control their adiposity efficiently, but the contribution of leptin to weight regulation in these animals remains unclear. To study the seasonality of overwintering raccoon dogs as well as the effects of fasting on them, serum leptin levels were investigated using a newly established canine leptin-specific enzyme-linked immunosorbent assay (ELISA) kit. Of the nine animals studied, five were fed and four were fasted (deprived of food for 2 months in winter). Blood samples and body fat weights were monitored once a month throughout the experimental period (July 2007-March 2008). Leptin concentrations obtained by ELISA were significantly higher than and had a positive correlation with those obtained by previously used multispecies radioimmunoassay (RIA) kits. Moreover, ELISA showed a clearer correlation between the body fat weight and leptin levels compared with RIA, suggesting the efficacy of canine leptin-specific ELISA kit for leptin estimation in raccoon dogs. Autumnal fattening was observed in both groups of animals, but the wintertime loss of adipose tissue was more obvious in the fasted group. Serum leptin concentrations determined by ELISA showed seasonal changes without significant differences between the fed and fasted animals. Therefore, high levels of leptin may be responsible for the suppression of feeding behavior in raccoon dogs before winter.  相似文献   

17.
The present paper describes (i) a hydrolysis technique with Pronase and leucine aminopeptidase using one rat thyroid gland, resulting in maximum release of thyroid hormones and minimum deiodination, and (ii) a simple and rapid procedure for thyroid hormone radioimmunoassays in thyroid hydrolysates using commercial kits intended for serum thyroid hormone determinations. The procedure is used to determine T4, T3, and rT3 concentrations and hormonal molar ratios in a thyroid gland from a male Wistar rat. The reliability of the method is also studied.  相似文献   

18.
目的:obestatin是新发现的与肥胖相关的多肽,本研究探讨幽门螺杆菌(H.pylori)对体重指数(BMI)~LM浆obestatin水平的影响。方法:于我院体检中心入选健康体检者205例,所有入选者无明确的胃肠疾病史,无心脏、肾脏、肝脏、甲状腺等疾病病史,无急性感染等其他可能影响激素水平的因素。幽门螺杆菌的感染依据l3C呼气实验及血清学指标联合判定。应用放射免疫分析法测定血浆obestatin及血清胰岛素水平。血清血脂水平采用自动生化分析仪进行酶法测定。胰岛素抵抗采用胰岛素抵抗指数(HOMA-IR)评定,HOMA—IR=空腹胰岛素(p~IU/ml)x空腹血糖(mmol/L)/22.5。结果:在我们的75例研究对象中H.pylori阳性的检出率为39%,幽门螺杆菌感染阳性者与阴性者相比,体重指数(BMI)差异并无统计学意义,血浆obestafin水平无显著性的差异。按体重指数进行分组,BMI〉24kg/m2组与BMI〈24kg/m2相比血浆obestatin水平明显下降。结论:H.pylori的感染状态并没有显著影响个体的循环obestatin水平。BMI对血浆obestatin水平有影响。  相似文献   

19.
Heikinheimo O  Ranta S  Grunberg S  Spitz IM 《Steroids》2000,65(10-11):831-836
Long-term administration of progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) has been proposed as a novel hormonal therapy for various hormone dependent maladies. We studied the long-term endocrine effects of mifepristone on the kinetics of estradiol (E(2)) and its precursors, and on gonadotropin levels in five postmenopausal women treated for unresectable meningioma with mifepristone [200 mg/day] for at least 15 months. Serum samples were analyzed for LH, FSH and SHBG with fluoroimmunoassay; androstenedione (A), testosterone (T), estrone (E(1)) and E(2) were measured with radioimmunoassay (RIA). Serum levels of mifepristone were measured using both RIA and high performance-liquid chromatography (HPLC). Serum levels (mean +/- SD) of LH and FSH were suppressed from pretreatment values of 32 +/- 16 and 65 +/- 30 IU/l to 13 +/- 7 and 33 +/- 16 IU/l at 6 months (P < 0.05), respectively. Serum (mean +/- SD) A, T, E(1), and E(2) were increased from initial values of 6.9 +/- 0.9 nmol/l, 1.2 +/- 0.3 nmol/l, 77 +/- 25 pmol/l, and 29 +/- 14 pmol/l to 6 month values of 13.1 +/- 5.6 nmol/l, 1.8 +/- 0.6 nmol/l, 178 +/- 60 pmol/l, and 45 +/- 22 pmol/l (n.s.). The correlation coefficients between the levels of A, T, E(1), and E(2) were statistically significant, whereas the ratios of T/A, E(1)/A, E(2)/E(1), and E(2)/T remained unchanged. The levels of SHBG remained stable, and ranged from 48 +/- 10 to 65 +/- 9 nmol/l (mean +/- SD). Thus, prolonged mifepristone treatment marginally increased the serum levels of A, T, E(1) and E(2). These effects of mifepristone are likely due to its antiglucocorticoid effect and thus increased secretion of adrenal A. Serum levels of LH and FSH declined. The serum levels of gonadotropins and those of T, E(1) and E(2) were inversely, yet significantly, correlated. Therefore the decrease in LH and FSH might reflect the slightly increased levels of T, E(1) and E(2). However, the lack of change in SHBG and the low E(2) levels suggest that enhanced systemic estrogen effects are unlikely during long-term mifepristone treatment.  相似文献   

20.
Correlations between thyroid hormone levels and body dimensions were investigated in a group of 124 premenopausal (ages 16–40 years) and 142 postmenopausal (ages 38–61 years) women from Vienna, Austria. Twenty-nine absolute body dimensions and thirteen anthropometric indices were correlated with the serum levels of thyroxine, triiodothyronine, thyroid-stimulating hormone, and thyroid-hormone-binding globulin as well as three hormone ratios (T3/T4, T4/TSH, T3/TSH). All hormones exhibited statistically significant correlations with 24 anthropometric variables and 11 indices. The correlations between thyroxine and triiodothyronine levels and the body measurements were predominantly positive in both proband groups. Higher thyroid hormone levels were associated positively with body dimensions, especially with the amount of subcutaneous fat tissue, in adult females independently of their menstrual status. The direction of the correlations between thyroid-stimulating hormone and body measures as well as anthropometric indices differed between the premenopausal and the postmenopausal women. While in premenopausal women mainly positive correlations between anthropometric characters and the level of thyroid stimulating hormone occur, in postmenopausal women most of these correlations are negative. This is probably due to the decrease of thyroid-stimulating hormone levels with increasing age, as well as with changes in body shape during the climacteric and after the menopause. © 1994 Wiley-Liss, Inc.  相似文献   

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