Immunoradiometric assay (IRMA) for human follicle stimulating hormone (FSH) and luteinizing hormone (LH) using common avidin solid phase

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).     

A Sensitive and Specific Immuradiometric Assay (IRMA) for Human Thyroid Stimulating Hormone

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.     

Enhancing effect of human thyroid stimulating hormone (hTSH) monoclonal antibody in the hTSH immunoradiometric assay (IRMA) system

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.     

Limitations of follicle-stimulating hormone in assessing menopause status: findings from the National Health and Nutrition Examination Survey (NHANES 1999-2000)*

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.     

Production and Characterization of Monoclonal Antibodies against Bovine Luteinizing Hormone

Five mouse hybridoma cell lines producing monoclonal antibody against bovine luteinizing hormone (LH) have been established and the respective antibodies characterized by radioimmunoassay, immunofluorescence and immunoelectrophoresis. All antibodies belong to the IgG class and bind to staphylococcus protein A. Intraspecies cross-reactivity studies revealed no reaction with bovine follicle stimulating hormone (FSH). However, all antibodies showed partial cross-reaction with bovine thyroid stimulating hormone (TSH) suggesting a close conformational similarity between bovine LH and TSH. Studies on interspecies cross-reactivity (rat and human) showed that three of these five antibodies strongly react with rat LH but not at all with either rat FSH or rat TSH thus representing monospecific reagents for investigations concerning LH in this species. One of these three antibodies also strongly binds to human LH and to the same extent to human chorionic gonadotropin (CG) but not to human FSH or TSH. It was concluded that at least three different epitopes on the bovine LH molecule are recognized and that they are located on the β-chain of the hormone.     

A sensitive and specific immunoradiometric assay (IRMA) for human thyroid stimulating hormone

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.     

Development of an immunoenzymometric assay (IEMA) for the estimation of human thyroid stimulating hormone (hTSH) in serum

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.     

FITC与抗-FITC系统检测促黄体生成素的应用评价

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.     

Development and Validation of a Simple,Sensitive, Second Antibody Format Enzyme Immunoassay (EIA) for LH Determination in Mithun (Bos Frontalis) Plasma

Abstract

The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin‐streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin‐peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20?µL mithun plasma. The LH standards ranging from 6.25?pg/well/20?µL to 400?pg/well/20?µL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25?pg/well LH, which corresponds to 0.31?ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100?pg/well/20?µL. Plasma volumes for the EIA viz. 10 and 20?µL did not influence the shape of standard curve even though a slight drop in the OD₄₅₀ was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10?µg i.v., with a synthetic analogue of GnRH (Buserelin‐Acetate, Intervet, India) and blood samples were collected at 15?min intervals using indwelling jugular catheter beginning 1?h prior to GnRH injection till 8?h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun.     

绝经前乳腺癌患者术后辅助化疗对血清性激素六项的影响

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。     

Genitourinary phenotype in XX patients with distal 9p monosomy

Although testicular development has been shown to be variably impaired in XY patients with distal 9p monosomy, ovarian and other genitourinary phenotype has poorly been studied in XX patients monosomic for the distal 9p region. Thus, we studied a 13-month-old infant with 46,XX,der(9)t(9;10)(p23;p13) (case 1) and an 11-year-old girl with 46,XX,der(9)t(9;16)(p23;q22) (case 2). Case 1 had primary hypogonadism (basal serum follicle stimulating hormone [FSH], 40.0 mIU/mL; leteinizing hormone [LH], 1.2 mIU/mL; estradiol [E2], <10 pg/mL), whereas case 2 had age-appropriate pubertal development (breast, Tanner stage 4; pubic hair, Tanner stage 3; menarche 11.7 years of age) and hormone values (FSH, 7.3 mIU/mL; LH, 6.7 mIU/mL; E2, 47 pg/mL). In addition, case 1 had hypoplastic labia majora, short distance between the vaginal orifice and the anus, and five renal cysts, and case 2 had anal atresia, short distance between the vaginal orifice and the anus, bilateral hydronephrosis of grade 3 with probable ureteropelvic junction stenosis, and renal dysfunction (serum creatinine, 1.52 mg/dL; urea nitrogen, 34.5mg/dL). Fluorescence in situ hybridization analysis for five regions and microsatellite analysis for 10 loci on 9p confirmed hemizygosity for the distal 9p region with the breakpoints between IFNA and D9S285 in case 1 and between D9S168 and D9S286 in case 2. The results, in conjunction with the previous data in XX patients with molecularly defined distal 9p monosomy, are consistent with the presence of a gene(s) involved in the development of indifferent gonad or subsequent ovarian differentiation in a approximately 11 Mb region distal to D9S168. In addition, it is possible that a gene(s) for anoperineal and renal development also maps distal to D9S168 and that for external genital development maps distal to D9S285 at the position approximately 16 Mb from the 9p telomere.     

Endocrinology: The combination of gonadotrophin-releasing hormone (GnRH) antagonist and pulsatile GnRH normalizes luteinizing hormone secretion in polycystic ovarian disease but fails to induce follicular maturation

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.     

TIME-RESOLVED IMMUNOFLUOROMETRY OF SERUM HTSH WITH ENHANCED SENSITIVITY

ABSTRACT

Sensitive TSH immunoassays offer a clear advance in discriminating the TSH concentrations in serum between hyperthyroid and euthyroid individuals; they have been proposed as the best single screening test for thyroid disorders. We have developed a highly sensitive serum TSH TRFIA based on DELFIA technology. Three monoclonal antibodies (McAbs) directed against different epitopes of the TSH molecule were involved in this assay, of which, one McAb was used to coat clear microwells, and the other two were biotinylated for signal generation after being bound by the europium labeled streptavidin. The europium label captured on the well surface was quantified by a routine dissociation-enhancement procedure. The fluorescence intensity was directly proportional to the serum hTSH concentration. The assay required two steps and could be completed within 5 h. The analytical sensitivity reached 0.002 mIU/L with a sample volume of 100 µL, the function sensitivity was 0.017 mIU/L. Measurements by the present method correlated well with that obtained by the ACS-180 chemi-luminescence immunoassay (CLIA). The discrimination of hyperthyroid patients from clinically euthyroid patients by the present method was much better than that by the routine IRMA.     

Monoclonal Antibodies Against Human Chorionic Gonadotropin (hCG): I. Production,Specificity, and Intramolecular Binding Sites*

ABSTRACT: Thirty-nine monoclonal antibody (MCA) producing hybridoma cell lines derived from fusions of mouse myeloma cells with spleen cells from mice immunized with human chorionic gonadotropin (hCG) have been established. Their products have been tested in radioimmunoassays using ¹²⁵I-labeled hCG, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), the alpha (α) and beta (β) subunits of hCG and LH, and the C-terminal peptide 109–145 (CTP) of CG. All MCA were, in addition, tested in indirect immunofluorescence (IIF) on paraffin sections of human pituitary glands. According to the intramolecular localization of the determinants recognized, three main groups of MCA can be distinguished: 1) MCA directed against epitopes on the β-chain (α-MCA), 2) MCA directed against (β-chain determinants (β-MCA), and 3) MCA reacting with a conformational determinant only present on the native hormone and not on either subunit (con-formational-MCA). All α-MCA cross-react with human LH, FSH, and TSH. The β-MCA do not react with FSH or TSH, but do react to a varying degree with LH. The conformational-MCA show no binding of labeled FSH or TSH and very little or no cross-reactivity with LH.     

Development and Application of a Sensitive,Second Antibody Format Enzymeimmunoassay (EIA) for Estimation of Plasma FSH in Mithun (Bos frontalis)

Mithun (Bos frontalis) is a semi-wild rare ruminant species. A simple sensitive enzymeimmunoassay suitable for assaying FSH in the blood plasma of mithun is not available which thereby limits our ability to understand this species reproductive processes. Therefore, the aim of this article was to develop a simple and sensitive enzymeimmunoassay (EIA) for estimation of FSH in mithun plasma and apply the assay to understand the estrous cycle and superovulatory process in this species. To accomplish this goal, biotinylated FSH was bridged between streptavidin-peroxidase and immobilized antiserum in a competitive assay. Forty microlitre mithun plasma was used directly in the EIA. The FSH standards were prepared in hormone free plasma and ranged from 5–1280 pg/well/40 μL. The sensitivity of EIA was 5 pg/well FSH, which corresponds to 0.125 ng/mL plasma and the 50% relative binding sensitivity was 90 pg/well/40 μL. Although the shape of the standard curve was not influenced by different plasma volumes viz. 40 and 80 μL, a slight drop in the OD₄₅₀ was observed with the increasing volume of plasma. Parallelism tests conducted between the endogenous mithun FSH and bovine FSH standards showed good homology between them. Plasma FSH estimated using the developed EIA and commercially available FSH EIA kit in the same samples were correlated (r = 0.98) and showed linearity. Both the Intra- and inter-assay CV were below 6%. Recovery of known concentrations of added FSH showed linearity (r = 0.99). The developed EIA was further validated biologically by estimating FSH in cyclic cows for the entire estrous cycle, in mithun heifers administered with GnRH analogues and in mithun cows during superovulatory treatment with FSH. In conclusion, the EIA developed for FSH determination in mithun blood plasma is simple and highly sensitive for estimation of mithun FSH in all physiological conditions.     

A NEW MODEL ELISA,BASED ON TWO MONOCLONAL ANTIBODIES,FOR QUANTIFICATION OF FATTY ACID SYNTHASE

ABSTRACT

A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54–121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 ± 1.81 ng/mL, 4.25 ± 2.14 ng/mL in women (n = 37) and 3.70 ± 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal–monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS–ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal–monoclonal FAS–ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.     

Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle stimulating hormone

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     

育龄期非妊娠妇女血清低水平β-HCG与LH、FSH的临床意义

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.     

FSH双标记液相IRMA的方法建立及实验研究

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。     

Sensitive ELISA for Determination of Serum E2 Using a New Tracer E2-Biotin

Abstract

A new tracer conjugate of E₂-Biotin, with different spacers, was synthesized at position 3 in the estradiol molecule for first time. Immunoreactivity of the tracer was determined by reacting with the anti-E₂ monoclonal antibody. The monoclonal antibodies raised against E₂ were characterized for its use in ELISA detection systems of serum E₂. The purified antibody has a high affinity and specificity for E₂. The antibody and tracer were used for establishing a competitive ELISA for estradiol (E₂). The experimental results showed that the dose-response curve of the assay covered a range of 33–20,000 pg/mL (n = 8). The detection limit is 28.3 pg/mL (S/N = 3). The intra- and inter-assay coefficients of variation for the assay of serum samples ranged from 5.7 to 13.2% and from 5.3 to 10.6%, respectively. Precoated microtiter plates were dried at 4°C and they were stable for up to 3 months.