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1.
Abstract

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with 125I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0–200 mIU/mL) and analytical recovery (87–110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).  相似文献   

2.
Different monoclonal antibodies, both commercial and indigenously produced, were evaluated in various combinations to optimize an immunoradiometric assay (IRMA) system for human thyroid stimulating hormone (hTSH). During these studies, it was observed that mixing one of the indigenously produced hTSH monoclonal antibody (2B11) in the hTSH IRMA system using Immunotech (Beckman Coulter, Czech Republic) kit reagents, led to an overall increase in the assay binding and sensitivity (from 0.025 mIU/L to 0.015mIU/L) of this IRMA system. This is not a general property of all monoclonal antibodies against hTSH. The mechanism for this enhancement can be attributed to the formation of a multicomponent complex.  相似文献   

3.
A liquid phase "two-site" immunoradiometric assay (IRMA) specific for human thyroid stimulating hormone (hTSH) is described. The assay is based on the simultaneous addition of affinity purified sheep anti hTSH IgG-I 125 and rabbit anti hTSH antiserum to standards and unknowns followed by 4h incubation at room temperature. The separation of free labelled sheep IgG-I125 from that bound to hTSH is achieved by the addition of sheep anti-rabbit IgG Fc fragment antiserum. The radiolabelled sheep anti-hTSH IgG-I 125 was pretreated with solid phase urinary postmenopausal gonadotropins to remove cross reaction with FSH and LH. The assay is specific for hTSH and no cross reaction with the other anterior pituitary glycoproteins or protein hormones has been found. In addition it is characterized by a wide operating range, rapid equilibration of reactants and high sensitivity (0.02 microU/ml). The precision of dose estimates was less than 10% between 0.25-2.5 microU/ml and less than 2.5% over the range 2.5-60 microU/ml.  相似文献   

4.
OBJECTIVE: We used data from the National Health and Nutrition Examination Survey (NHANES 1999-2000) to: establish new population-based estimates for follicle-stimulating hormone (FSH) and luteinizing hormone (LH); identify factors associated with FSH; and assess its efficacy in distinguishing among women in the reproductive, menopause transition, and postmenopausal stages. DESIGN: Nationally representative sample of 576 women aged 35 to 60 years examined during NHANES 1999-2000. RESULTS: Levels of FSH and LH increased significantly with reproductive stage. (Geometric mean FSH levels for successive stages: reproductive, 7.0 mIU/mL, SE 0.4; menopause transition, 21.9 mIU/mL, SE 3.7; and postmenopause, 45.7 mIU/mL, SE 4.3). There was considerable overlap, however, among distributions of FSH by stage. Only age and reproductive stage were significantly associated with FSH in multivariable analysis. FSH cutoff points between the reproductive and menopause transition stages [FSH = 13 mIU/mL, sensitivity 67.4% (95% CI 50.0-81.1), specificity 88.1% (95% CI 81.1-92.8)] and between the menopause transition and postmenopause stages [FSH = 45 mIU/mL, sensitivity 73.6% (95% CI 60.1-83.7), specificity 70.6% (95% CI 52.4-84.0)] were neither sensitive nor very specific. CONCLUSIONS: Age and reproductive stage are the most important determinants of FSH levels in US women; however, FSH by itself has limited utility in distinguishing among women in different reproductive stages.  相似文献   

5.
A liquid phase “two-site” immunoradiometric assay (IRMA) specific for human thyroid stimulating hormone (hTSH) is described. The assay is based on the simultaneous addition of affinity purified sheep anti hTSH IgG-I125 and rabbit anti hTSH antiserum to standards and unknowns followed by 4h incubation at room temperature. The separation of free labelled sheep IgG-I125 from that bound to hTSH is achieved by the addition of sheep anti-rabbit IgG Fc fragment antiserum. The radiolabelled sheep anti-hTSH IgG-I125 was pretreated with solid phase urinary postmenopausal gonadotropins to remove cross reaction with FSH and LH. The assay is specific for hTSH and no cross reaction with the other anterior pituitary glycoproteins or protein hormones has been found. In addition it is characterized by a wide operating range, rapid equilibration of reactants and high sensitivity (0.02 μU/ml). The precision of dose estimates was < 10% between 0.25–2.5 μU/ml and < 2.5% over the range 2.5–60μU/ml.  相似文献   

6.
A sensitive immunoenzymometric assay (IEMA) of serum thyrotropin (hTSH) was developed using anti-hTSH rabbit polyclonal antibody and anti-hTSH in-house monoclonal antibody with a sensitivity of 0.12 mIU/L. Serum samples were incubated in ELISA wells precoated with polyclonal antibody. The hTSH bound to the wells was incubated with monoclonal antibody (detector antibody) and further with goat anti-mouse antibody-horse radish peroxidase (GAM-HRP), which obviates the need to label the detector antibody. The assay was validated by recovery, linearity, and cross-reactivity experiments with a working assay range of 0.15 to 100 mIU/L and <10% coefficient of variation (CV) for both intra- and interassay. Good correlations were obtained when compared with Immunotech hTSH IRMA (r = 0.971, n = 35). This in-house ELISA can be used as an initial screening test for thyroid dysfunction.  相似文献   

7.
目的 使用抗-异硫氰酸荧光素(FITC)固相包被板建立促黄体生成素(LH)的酶联免疫(ELISA)检测方法.方法用FITC及辣根过氧化物酶(HRP)分别标记两株抗-LH单克隆抗体,建立一步检测LH的ELISA检测方法,并与经典的双抗体夹心法进行了方法学对比评价.结果 FITC与抗-FITC系统检测LH,在2~50mIU/mL的校准曲线范围内相关系数0.9968,分析内精密度为7.6%,分析间精密度为7.02%,热稳定性下降18.5%,与双抗体夹心法相当,空白检测限为0.15mIU/mL,优于双抗体夹心法,钩状效应(HOOK效应)比双抗体夹心法差.与贝克曼检测结果回归方程Y=0.970X+0.614,相关系数r=0.975.结论 成功建立基于FITC与抗-FITC系统的ELISA检测方法,与双抗体夹心法相比,两种方法均能满足临床检测的需要.  相似文献   

8.
To evaluate the role of altered luteinizing hormone (LH) releasein the mechanism of polycystic ovarian disease (PCOD) anovulation,we have co-administered a gonadotrophin-releasing hormone (GnRH)antagonist and pulsatile GnRH therapy to two clomiphene citrate-resistantPCOD patients. The aim was to correct their inappropriate gonadotrophinsecretion. Nal-Glu was administered s.c. every 72 h to bothsubjects for 3 weeks. On day 7 after commencing the study, intravenouspulsatile GnRH therapy was initiated (10 ug/ pulse) every 90min for 15 days to both subjects. In one subject, Nal-Glu treatmentwas continued and the GnRH dose was increased to 20 ug/pulsefor 10 additional days. Prior to Nal-Glu, mean serum LH levelswere 10.4 ±1.6 and 9.3 ± 1.3 mlU/ml (mean ±SEM) and mean interpulse intervals were 67.1 and 60 min in patients1 and 2, respectively. Mean serum FSH levels were 4.9 ±0.4 and 4.2 ± 0.2 mIU/ml for patients 1 and 2, respectively.LH pulsatility was abolished following Nal-Glu, mean serum LHdecreased to 1.1 ± 0.1 and 1.3 ± 0.5 mIU/ml andmean FSH to 1.8 ± 0.1 and 2 ± 0.1 mIU/ml in the two subjects. On the 4th day ofthe combined therapy, mean serum LH increased to 5.4 ±1.3 and 3.9 ± 0.9 mIU/ml with a mean interpulse intervalof 72 and 80 min, respectively. Mean FSH levels increased to3 ± 0.1 and 2.8 ± 0.1 mIU/ml, respectively andto 5.5 ± 0.2 mIU/ml after the GnRH dose was increasedin patient 2. Testosterone levels decreased but remained abovethe normal range following combined therapy. Levels increasedto the pretreatment values after augmentation of the GnRH dosein subject 2. Oestradiol levels remained <50 pg/ml and nofollicular development was observed on vaginal endosonography.Failure to obtain an ovarian response in PCOD after re-establishmentof improved pituitary gonadotrophin release may reflect thepresence of an inherent ovarian defect in PCOD patients.  相似文献   

9.
An antiserum to follicle stimulating hormone (FSH) obtained as a gift from National Institute of Health (NIH), U.S.A. could not be adsorbed on microtitre ELISA plates, although two other FSH antisera raised in authors' laboratory could be adsorbed. A good precision profile for the FSH assay using these three antisera could be achieved with only one separation system viz. solid phase anti rabbit gamma globulin (ARGG), out of the five separation systems tried. The study suggests that a few antisera used for radio-immunoassay (RIA) purposes may not by themselves get adsorbed on plastic plates. However, they could be effectively used for ELISA purposes using solid phase second antibody.  相似文献   

10.
The present study examined the effects of the ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) on granulosa cell proliferation and oestradiol-17 beta secretion. For these studies, ovarian segments from either immature rats or those primed with pregnant mares serum gonadotrophin (PMSG) were incubated for 5 h with [3H]thymidine and FSH (0-100 mIU/ml) with or without equivalent doses of LH. After incubation, granulosa cells were isolated and their mitotic activity estimated by determining the amount of [3H]thymidine incorporated into the DNA. The amount of oestradiol secreted into the media was measured by radioimmunoassay. Compared to granulosa cells from immature ovaries, granulosa cells from PMSG-primed ovaries required significantly less FSH to stimulate incorporation of [3H]thymidine, had a 9-fold higher basal level of oestradiol production and increased oestradiol secretion in response to gonadotrophins. At pharmacological serum levels (10-20 mIU of total gonadotrophin), FSH:LH ratios of less than or equal to 2 increased oestradiol secretion from PMSG-primed ovaries but did not increase the rate of [3H]thymidine incorporation. Conversely, FSH:LH ratios of greater than or equal to 3 stimulated [3H]thymidine incorporation without altering oestradiol secretion. These data demonstrate that granulosa cells of immature follicles not secreting oestradiol are relatively unresponsive to gonadotrophins at any dose tested. Once the capacity for oestradiol secretion develops, then both the dose and ratio of FSH and LH play major roles in determining whether the follicle will grow or secrete oestradiol.  相似文献   

11.
Detecting pre-ovulatory luteinizing hormone surges in urine   总被引:2,自引:1,他引:2  
The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.   相似文献   

12.
Parenteral administration of follicle stimulating hormone (FSH) has been shown to lower luteinizing hormone (LH) concentrations in women undergoing ovulation induction. This study was designed to explore the physiological mechanism of this effect. Seven healthy women were recruited into a double-blind placebo-controlled study. LH secretion, after the administration of variable i.v. boluses (37.5, 75 and 150 IU) of recombinant FSH (Gonal-F), was evaluated. LH was measured at 10 min intervals for 2 h before and 4 h after the FSH/placebo infusion. LH pulse frequency and amplitude were evaluated and there was no significant difference between control and trial cycles for each subject. A linear regression analysis revealed that in the group receiving 150 IU FSH, the mean plasma LH concentration decreased significantly due to a reduction tonic LH secretion. This could be a result of the suppression of secretion or an alteration of clearance. This decrease was not seen in the other dosage groups, revealing that above a dosage threshold, FSH reduced non-pulsatile LH secretion. Therefore the effect of FSH in this study exposed the likely presence of two components of LH concentration: an FSH-sensitive, non-pulsatile tonic secretion and a gonadotrophin-releasing hormone-stimulated, pulsatile release that is unaffected by FSH. Although an indirect effect involving ovarian regulation is not excluded, the rapidity of the effect suggests that FSH acts directly on the pituitary gland.   相似文献   

13.
OBJECTIVE: This study describes age-related changes in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a 5-year prospective study of reproductive aging. DESIGN: Participants (n = 156 college-educated, white, US women; 25 to 58 y) were recruited from the TREMIN Research Program on Women's Health. They collected daily urine specimens for 6 months in each of 5 consecutive years. Specimens were assayed for LH and FSH. Aggregate changes were calculated in LH and FSH with age, and multilevel models were used to estimate individual hormone trajectories and within-woman and between-woman variances by age. RESULTS: Aggregate LH levels increased beginning after age 45; FSH increased at all ages, accelerating after age 45. Individual-level patterns with age included the following: reproductive-age LH and FSH levels, with increasing FSH and increasing or decreasing LH (ages 20 to 49); rapidly increasing LH and FSH (ages 40 to 59); and increasing or steady postmenopausal LH and FSH (ages 46 to 62). FSH levels were consistently high in the latter category, but LH levels overlapped with levels found in younger women (<45 y). Individual LH patterns showed more variability (5% to 35% of total variance) than FSH (3% to 22% of total variance). Both hormones had relatively low variation within individuals compared with between-woman differences (65% to 97% of total variance). CONCLUSIONS: Aggregate-level data do not reflect differences across women and oversimplify the age-related increases and variability in LH and FSH. Individual FSH levels are not distinguishable from reproductive-age levels until after rapid perimenopausal increases in FSH occur; individuals vary in whether their postmenopausal LH levels are distinguishable from reproductive-age levels.  相似文献   

14.
We have developed a sensitive, non-competitive, two-monoclonal antibody, sandwich-type or immunoradiometric assay for human chorionic gonadotropin (hCG) which shows no cross-reaction with the free beta chain of hCG nor with human luteinizing hormone (LH). In the assay procedure, two, highly selected monoclonal antibodies reacted in solution with hCG to be quantified. One antibody was covalently conjugated to biotin. This antibody was specific for the beta subunit of hCG, and showed no reaction with LH nor the alpha subunit. The second antibody was labelled with 125I and was specific for intact hCG and LH, showing no cross-reaction with beta hCG nor the alpha subunit. The separation system was a polystyrene ball conjugated with biotin. This ball bound via an avidin bridge the monoclonal 'sandwich' containing hCG. Counts per minute bound to the ball were directly proportional to the amount of hCG present. The assay was specific for whole hCG and showed no reaction with beta hCG, beta LH, intact LH nor the free alpha subunit. Sensitivity was adequate to detect 'hCG-like' material in all post menopausal women and, when single samples were obtained, in over 2/3 of normal men. When multiple samples were obtained, 'hCG-like' material was detectable in all eugonadal adults studied.  相似文献   

15.
目的:探讨磁性微粒子双标记IRMA检测FSH的临床应用价值及其重要意义。方法:利用^125Ⅰ和异硫氰酸荧光素(FITC)分别标记的单克隆抗体(McAb)与抗原进行夹心反应,然后加入抗荧光素抗体包被的磁性微粒进行沉淀分离。结果:检测样品值两法高度相关(r〉0.9900),双标记液相IRMA法批间变异CV(n=10)5.8%,包被管固相法批间变异CV(n=10)8.5%,两法差异显著。结论:双标记液相IRMA技术是一种快速有效、灵敏度高、特异性好的定量检测方法。  相似文献   

16.
In the present study, we analysed and compared the relativein-vitro biological activity of the various intrapituitary humanfollicle stimulating hormone (FSH) isoforms employing two differentbioassay systems. FSH was fractionated by chromatofocusing (pHrange 7.10 to <3.80) and the several isoforms isolated werequantified at multiple dose levels by three highly specificimmunoassay systems: radioimmunoassay (RIA), enzyme-immunoassay(EIA) and immunoradiometric assay (IRMA), as well as by twoin-vitro bioassays, one that measures the amount of oestrogenproduced by rat granulosa cells in culture and the other thatdetermines the amount of cAMP produced by a human fetal cellline (293) expressing the recombinant human FSH receptor. Therelative in-vitro biological activity of each FSH isoform, expressedas the bioassay/ immunoassay (B/I) activity ratio (B/RIA, B/EIAand B/IRMA ratios) varied with its elution pH value. Regardlessof the immunoassay or bioassay method employed, less acidicFSH isoforms exhibited higher B/l ratios than their more acidiccounterparts (B/RIA, B/EIA and B/IRMA ratios for isoforms withelution pH values >4.5 = 1.05 ± 0.13, 0.99 ±0.10 and 1.15 ± 0.08 (rat oestrogen bioassay), and 2.75± 0.34, 2.20 ± 0.25 and 2.96 ± 0.35 (humancAMP production bioassay) respectively. Ratios for isoformswith pH values <4.5 = 0.71 ± 0.06, 0.47 ± 0.05and 0.63 ± 0.06 (rat oestrogen assay), and 1.80 ±0.26, 1.10 ± 0.09 and 1.44 ± 0.13 (cAMP assay)respectively (P<0.05 for isoforms with pH <4.5 comparedwith those isoforms with pH >4.5)]. Furthermore, statisticallysignificant direct relationships between the B/RIA, B/EIA andB/IRMA ratios and the elution pH value of each isoform was identifiedby regression analysis [rat assay: r = 0.844, 0.800 and 0.780(P<0.01); human assay: r = 0.730, 0.845 and 0.821 (P<0.01),for their corresponding B/RIA, B/EIA and B/IRMA ratios respectively].The finding of significant differences in relative in-vitrobiological potency among the various intrapituitary FSH isoformsstrongly suggests that the shifts towards the production andsecretion of more basic or acidic FSH molecules occurring incertain specific physiological conditions (e.g. puberty andmenstrual cycle), may represent an important mechanism throughwhich the anterior pituitary regulates gonadal function. follicle stimulating hormone/FSH bioactivity/FSH glycoforms/granulosa cells/recombinant FSH receptor  相似文献   

17.
The effects were studied of follicle stimulating hormone (FSH)-inducedproduction of gonadotrophin surge-inhibiting factor (GnSIF)on three phases of the pituitary responsiveness to gonadotrophinreleasing hormone (GnRH): the unprimed, primed and desensitizedphases. Rats were injected with FSH on two occasions duringthe oestrous cycle. Spontaneous luteinizing hormone (LH) surgeswere measured as well as GnRH-induced LH surges on the day ofpro-oestrus during infusions with 100–4000 pmol GnRH/rat/10h, in phenobarbital blocked rats. The spontaneous LH surgeswere attenuated or completely inhibited by the FSH treatment.FSH suppresses and prolongs the unprimed LH response and delaysGnRH self-priming, especially during infusions with low concentrationsof GnRH. This treatment does not affect the total LH response(area under curve) to the highest concentrations of GnRH andafter ovariectomy. On the other hand, this response is suppressedduring infusions with the lower concentrations of GnRH. Hence,FSH, via GnSIF, delays maximal priming of the LH response toGnRH, whereas the suppression of LH release is a consequenceof the GnRH-induced progressed state of desensitization. Theinconsistent effects of FSH on the mid-cycle LH surges are explainedas a result of the interaction between the relative strengthsof GnRH and GnSIF.  相似文献   

18.
The role of gamma-aminobutyric acid (GABA) in the regulation of gonadotropin secretion in renal failure, was studied in 5 patients with chronic renal failure who were on maintenance dialysis. Di-n-propylacetic acid (valproic acid-VA), a GABA-transaminase inhibitor which has been shown to increase brain GABA levels, was used in the study. VA produced no significant change in the basal serum LH and FSH concentrations, or in E2 or T concentrations in the renal failure patients, or the E2 and P concentrations in normal controls, but augmented the delta LH (maximum increment above baseline) and delta FSH response to LH-RH. delta LH rose from 30.4 +/- 12.7 mIU/ml (mean +/- SD) to 41.1 +/- 16.8 mIU/ml (p less than 0.01) after VA, while delta FSH rose from 2.8 +/- 1.8 mIU/ml to 3.8 +/- 1.6 mIU/ml (p less than 0.05). The findings support a modulatory role for GABA in gonadotropin secretion in chronic renal failure.  相似文献   

19.
目的观察绝经前乳腺癌患者术后辅助化疗对其性激素6项的影响,为临床早期评价化疗导致卵巢损伤提供检验依据。方法应用回顾性分析及统计学方法,分析39例绝经前乳腺癌患者性激素6项化疗前和化疗后各时期的水平变化。结果化疗后各周期性激素6项与化疗前比较发现:FSH、LH在第一次化疗后就开始升高,FSH、LH在第二次化疗后结果分别为:39.9(9.19~102.1)mIU/mL,14.8(3.12~42.1)mIU/mL,与化疗前7.67(3.04~31.7)mIU/mL,4.31(1.91~22.8)mIU/mL比较差异有统计学意义,P<0.01,并随着化疗周期的增加持续升高并维持在较高水平;E2和P在第一次化疗后开始降低,E2在第二次化疗为:27.48(8.09~117.1)pg/mL与化疗前的51.1(15.38~363.56)pg/mL比较差异有统计学意义,P<0.01;P第三次化疗后为0.61(0.11~1.44)ng/mL与化疗前的1.57(0.27~23.2)ng/mL比较差异有统计学意义,P<0.01,并随着化疗周期的增加持续降低并维持在较低水平;T和PRL则在化疗前后及各化疗周期水平变化不明显,差异无统计学意义,P>0.05。结论绝经期前乳腺癌患者在化疗后,血清FSH、LH、E2、P均有显著变化,可暂将血清E2<27.48 pg/mL,FSH>39.9 mIU/mL,LH>14.8 mIU/mL,P<0.61 ng/mL作为判断化疗后卵巢损伤的启动点,有一定的临床价值。  相似文献   

20.
目的 探讨育龄期非妊娠妇女血清中检测出的低水平(β-HCG>0.10且<5.30mIU/mL)β-HCG以及LH、FSH的水平对临床的诊断意义.方法 通过对84例实验组血清中检出低水平β-HCG的育龄期非妊娠妇女及80例对照组(β-HCG <0.10mIU/mL)血清中LH、FSH、E2含量检测.结果 所选取研究对象的年龄对研究无影响;育龄期非妊娠妇女血清LH、FSH均较对照组高,其差异有统计学意义(P<0.05).结论 当育龄期非妊娠妇女血清检测到低水平β-HCG时,进一步检测其血清FSH和LH的浓度,可以对停经与卵巢功能相关性评价提供诊断参考.  相似文献   

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