双峰驼血浆促卵泡素的放射免疫分析法

用单峰驼促卵泡素标准品（ＣａｍＦＳＨ）,ｈＦＳＨ抗血清和＾１２５Ｉ－ｈＦＳＨ建立了测定双峰驼血浆ＦＳＨ的放射免疫分析方法,并通过一系列实验证明,该方法可以用于测定双峰驼血浆ＦＳＨ,是研究双峰驼生殖内分泌学的可靠手段之一。     

兔抗五种鱼免疫球蛋白抗体的制备和HRP标记

目的制备兔抗鱼类免疫球蛋白抗体并进行辣根过氧化酶标记,为鱼类血清学检测系统的建立提供工具。方法利用proteinA亲和层析的方法纯化鱼血清免疫球蛋白,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗鱼免疫球蛋白的抗体,采用简易过碘酸钠法对纯化的兔抗鱼免疫球蛋白的抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting来考察标记抗体与其他常见鱼类血清蛋白间的交叉反应。结果纯化了鳙、鲤、乌鳢、黄鳝、鲈五种鱼血清免疫球蛋白,免疫双扩散法测定兔抗这五种鱼免疫球蛋白的抗血清效价均达到1∶32,并对纯化的兔抗鳙、鲤、乌鳢、黄鳝、鲈五种鱼类免疫球蛋白的抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶10000左右,Western blots显示标记抗体与部分其他鱼类免疫球蛋白之间存在不同程度的交叉反应。结论成功制备了HRP标记的兔抗鱼类免疫球蛋白抗体,为鱼类血清学检测体系的建立提供了工具。     

用放射免疫测定法研究性类固醇激素和棉酚对大白鼠垂体LH分泌的影响

用兔抗羊 LH 血清和放射性碘标记的羊 LH 建立了大白鼠血清中 LH 的双抗体放射免疫测定法。在这异源的系统中羊 LH 和大鼠 LH 的剂量反应曲线是平行的。当用羊LH_(2-2-1)作参考标准时测量的灵敏度为0.1ng/管。当用大鼠 LH-RP-1作参考标准时测量的灵敏度为10ng/管。大鼠的 FSH 没有交叉反应,所以 FSH 不会干扰血清 LH 的测定。用这个系统我们研究了去卵巢以及去卵巢后给予孕酮和巳烯雌酚以后大鼠血清中LH 水平的变化。结果表明性类固醇激素对大鼠垂体 LH 的分泌具有一个负反馈的调节机制。本文中还研究了棉酚对大鼠 LH 分泌的影响。我们发现成年雄鼠服用棉酚以后血浆 LH 的水平以及垂体对 LRH 的反应均无显著的变化。结果表明新的男性抗生育药物——棉酚似乎不影响垂体的 LH 分泌功能。     

川金丝猴(Rhinopithecus roxellanae)月经周期促黄体素(LH)的分泌水平

本工作用改进的LH体外生物测定法,测定了一只川金丝猴两个月经周期的晨尿LH含量。结果表明LH峰分别出现在各月经周期的第16天和第11天。LH峰的平均水平为100.0±9.0 miu/ml尿[以人促黄体素(hLH)69/104作为标准],是滤泡期LH平均水平的6倍,黄体期LH平均水平的4倍。提示川金丝猴月经周期LH的分泌情况与人和猕猴的极为相似。     

乌鳢血清免疫球蛋白IgM的分离纯化及其兔抗血清的制备

目的 分离纯化乌鳢血清免疫球蛋白,并制备其兔抗血清。方法 用Protein A亲和层析的方法纯化乌鳢血清免疫球蛋白,通过SDS-聚丙烯酰胺凝胶电泳检测蛋白的纯度,测定其重链、轻链的分子量,免疫大耳白兔制备抗血清,利用免疫双扩散检测抗血清的效价。结果 纯化了乌鳢血清免疫球蛋白,SDS-PAGE测定其重链和轻链的相对分子质量分别为78×10^3和27×10^3左右,免疫双扩散法测定兔抗乌鳢免疫球蛋白抗血清效价为1∶32。结论 成功纯化了乌鳢免疫球蛋白,制备了兔抗乌鳢IgM抗血清,为研究乌鳢的免疫机制、建立乌鳢的血清学检测系统奠定了基础。     

LH与hCG对昆明小鼠卵母细胞体外成熟的影响

目的研究促黄体素（LH）、人绒毛膜促性腺激素（hCG）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得未成熟卵母细胞,分别在含不同浓度的LH和hCG的成熟液中,或将LH和hCG以不同的浓度组合加入到成熟液,进行体外成熟。结果经15.16h的成熟培养,5个浓度LH组中的极体率均高于对照组,其中200IU/mL组显著高于50IU/mL、400IU/mL、300IU/mL组和对照组（P〈0.05）;5个浓度hCG组的极体率与对照组极体率无显著差异（P〉0.05）;协同组中15IU/mL hCG＋200IU/mL LH组的极体率显著高于对照组和其它各处理组。结论LH对小鼠卵母细胞的体外成熟有一定的促进作用。     

2种β2-受体激动剂抗血清的比较研究

目的:通过比较分析,为β2-受体激动剂的快速免疫学检测方法及最佳免疫抗血清的选择提供依据。方法:分别以偶氮化法和碳二亚胺法将β2-受体激动剂克伦特罗(CL)和沙丁胺醇(Sal)连接到牛血清白蛋白和卵清蛋白上,免疫家兔;4次免疫后,采血制备抗CL和抗Sal的抗血清;用间接ELISA检测抗血清效价,用间接抑制ELISA检测抗血清交叉反应。结果:为抗CL抗血清的效价为1∶2560,抗Sal抗血清的效价为1∶5120。抗CL抗血清对Sal有0.088%的交叉反应,而抗Sal抗血清对CL有200%的交叉反应;就对CL的抑制而言,抗Sal血清比抗CL血清更敏感。结论:在检测β2-受体激动剂CL时,Sal完全抗原可能是制备抗血清的最佳抗原。     

草鱼生长激素非竞争式酶联免疫吸附测定法的建立及鉴定

应用草鱼生长激素（ｇｃＧＨ）单克隆抗体及多价兔抗血清建立了草鱼ＧＨ非竞争式酶’联免疫吸附测定ＥＬＩＳＡ系统。用正辛酸法对腹水单抗进行了分离纯化,获得了高纯度的单抗制备物。聚丙烯酸胺凝胶电泳表明纯化的单抗由分子量分别为５５ｋＤ和２５ｋＤ的两条蛋白带组成。用纯化单抗铺底,用兔抗血清作后续抗体建立了一种测定草鱼ＧＨ的非竞争式双抗夹心ＥＬＩＳＡ方法。交叉试验表明该测定系统只与草鱼ＧＨ和基因重组鲤生长激素（ｒｃＧＨ）具有剂量依存的结合反应,而与大马哈鱼生长激素（ｓＧＨ）、牛生长激素（ｂＧＨ）、大马哈鱼促性腺激素（ｓＧｔＨ）、及黑鲢促性腺激素（ｂｓｃＧｔＨ）等均无交叉反应。该 ＥＬＩＳＡ方法的灵敏度可达０．８ｎｇ／ｍｌ,组内变异系数为 ５．９ ％,组间变异系数为７．６％,回收率达９０％以上。初步应用表明,鲤和团头鲂垂体抽提液、草鱼血清、鲤血清及鲫血清在该测定系统中有剂量依存的反应曲线,而大口鲶、黄颡鱼、中华鲟及黄鳝鱼垂体抽提液及大口鲶、胡子鲶和罗非鱼血清在该测定系统中没有交叉反应。     

蝙蝠血清IgG的纯化及兔抗蝙蝠IgG酶标抗体的制备

目的纯化蝙蝠血清IgG,制备兔抗蝙蝠IgG酶标抗体。方法采用亲和层析纯化法纯化蝙蝠血清IgG,SDS-PAGE电泳鉴定蝙蝠IgG纯度。免疫大白兔制备兔抗蝙蝠IgG抗血清,免疫双扩散法测定抗血清效价,亲和层析纯化法纯化抗血清IgG。用改良过碘酸钠标记法制备兔抗蝙蝠IgG酶标抗体,直接ELISA和Western blot法对兔抗蝙蝠IgG酶标抗体进行工作浓度测定。结果纯化的蝙蝠血清IgG,其SDS-PAGE测定纯度大于95%;免疫大白兔所制备的抗血清免疫双扩散效价为1∶64;用改良过碘酸钠标记法制备兔抗蝙蝠IgG酶标抗体,其直接ELISA和Western blot工作浓度分别为1∶12800和大于1∶2000。结论制备了蝙蝠血清IgG的抗血清和酶标抗体,为蝙蝠的血清学检测体系提供了技术和资源储备。     

单克隆抗体与抗血清联合应用建立血清CMLC测定方法的研究

单克隆抗体(McAb)和抗血清各有特点。本文提纯人心肌肌球蛋白轻链(CMLC)并制备其单克隆抗体和抗CMLC兔血清(下称抗血清),试建立测定血清CMLC的酶联免疫(ELISA)方法。通过比较,McAb和抗血清联合应用可提高测定方法的灵敏度和特异性;并对各反应试剂的工作浓度进行确定,建立了McAb(1C_(11)-D_7株)为铺底抗体,抗血清为覆盖抗体,碱性磷酸酶标记的羊抗兔IgG为第三抗体的三抗体酶联免疫夹心测定血清CMLC的方法(MP-ELISA)。血清CMLC最小可测浓度为2.5ng/mL。     

Changes in testicular hCG binding and Leydig cell function in rats throughout life

The age dependence of Leydig cell function was investigated in rats from prepuberty (15 days) to senescence (39 months). Serum LH, serum and testicular testosterone were measured by radioimmunoassay. The binding capacity and affinity of LH/hCG receptors were determined by a radioligand receptor assay (hCG/Leydig cells) using 125I-hCG labelled by the lactoperoxidase method. Separation of bound and free 125I and simultaneous concentrations of 125I-hCG was achieved by vacuum ultrafiltration. The biochemical integrity of 125I-hCG tracer was ascertained by various chromatographic procedures. The highest hCG-finding and highest serum LH levels were found during puberty. Serum and testicular testosterone concentrations, however, were maximal in early adulthood. From this period onwards to late senescence hCG-binding changed only slightly, while serum LH and testosterone levels decreased significantly towards late senescence. The study shows that, although hCG binding to the Leydig cell changes characteristically during development, it is minimally affected by aging and cannot therefore be responsible for the reduced androgen biosynthesis in senescence.     

Radioimmunoassay system for norethisterone using 3H- and 125I-labelled radioligands.

A radioimmunoassay procedure is outlined for norethisterone, a synthetic progestagen. This assay uses both tritiated and iodine-125 labelled radioligands and may serve as a model for assays of synthetic steroids for which no tritiated radioligand exists. Male volunteers took a single oral dose of 10 mg of norethisterone acetate (SH 420). Plasma hormone levels were then measured at various time intervals. The degree of binding of iodine-125 labelled radioligand to antiserum even at low serum dilution was always greater than 80%. Using antinorethisterone-11 alpha-BSA serum, triated norethisterone and norethisterone-3-OCMO-iodine -125-iodohistamine radioligands give comparable results of adequate specificity, precision, accuracy and sensitivity when used to analyze crude ether extracts of the plasma samples. The chromatographic step is unnecessary for specific analysis. Iodine-125 labelled ligands may be useful for the determination of other synthetic steroids.     

125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.     

Mating-induced luteinizing hormone surge and ovulation in the female camel (Camelus dromedarius)

Blood samples were collected after mating from four female one-humped camels every 10 min for 9-12 h. Luteinizing hormone (LH) was quantified in plasma by radioimmunoassay using antibovine LH. Of the seven observed matings, five were followed by a release of LH, and three by an ovulation (indicated by a subsequent secretion of progesterone). LH levels at the time of mating ranged from 0.7 to 3 ng/ml. When an LH response occurred, the levels increased 1 h after mating and reached a maximum in 2-3 h (ranging from 2.9 to 19.1 ng/ml). A decrease in LH was observed starting 6 h after mating and lasting for 6 h. These results are in agreement with a coitus-induced mechanism of ovulation in the one-humped camel (Camelus dromedarius). They confirm and extend the observations reported in the bactrian camel (Camelus bactrianus).     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Comparisons of gonadotropin binding sites and progesterone concentrations among the corpora lutea of individual pigs

In polyovular species, it is unclear whether the characteristics of each individual corpus luteum (CL), such as mass, progesterone concentration and receptors for luteinizing hormone (LH), are representative of those of its cohorts during the ovarian cycle. The current study was performed 1) to characterize the conditions for estimation of binding parameters for LH receptors in porcine CL, and 2) to compare LH binding sites, luteal progesterone concentrations and luteal masses among CL of ovaries within individual pigs. Gonadotropin binding sites in porcine CL were characterized via specific binding of 125I-human (h) LH to 20,000 X g particulate fractions of luteal tissue. Specific binding was directly proportional to tissue content and was detectable at the lowest content tested (0.5 mg tissue equivalents/tube). Specific uptake of 0.25 ng LH by 5.0 mg tissue equivalents was time- and temperature-dependent; steady-state binding was achieved within 20 h at 37 and 25 degrees C. Binding of LH after 20 h incubation at 37 degrees C (4718 +/- 192 cpm, means +/- SEM) and 25 degrees C (4112 +/- 340 cpm) was greater than that at 4 degrees C (1930 +/- 5 cpm, P less than 0.01). Luteal particulates from individual CL of ovaries collected from four mature nonpregnant pigs (13-23 CL/pig) were incubated with eight concentrations of 125I-hLH. Steady-state binding depended upon hormone concentration until reaching saturation at 2.5 ng 125I-hLH/tube. Scatchard analyses yielded linear plots. Binding capacities for LH ranged among pigs from 0.71 +/- 0.03 to 3.69 +/- 0.13 fmol/mg CL equivalents and receptor affinities (Kd) ranged from 0.92 +/- 0.05 to 4.89 +/- 0.41 X 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I125LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L−1. The intra-assay coefficient of variation was 37% at 1 μg L−1, declined to 15% at 4 pg L−1 and was below 6% for concentrations up to 32 μg L−1. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L−1), 16% (7.1 μg L−1) and 9.8% (19 μg L−1). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L−1. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

125I-radioimmunoassay for antipyrine

A radioimmunoassay (RIA) for the determination of antipyrine directly in plasma and saliva has been developed using 4-¹²⁵I-iodoantipyrine as the radioligand. The method showed excellent agreement (r=0.98) with a recently reported RIA for antipyrine using ³H-antipyrine as the radioligand. The interassay coefficient of variation for the ¹²⁵I-RIA did not exceed 7.6% and the mean recovery of antipyrine added to plasma or saliva was 101±1.73 (S.E.) over a range of 1.5 to 30 μg/ml. By virtue of its simplicity and low cost, the ¹²⁵I-RIA for antipyrine offers an attractive method for the routine determination of antipyrine levels and subsequent calculation of its half-life in man.