抗人绒毛膜促性腺激素单克隆抗体的制备与鉴定

利用杂交瘤技术制备了11株抗人绒毛膜促性腺激素(hCG)的单克隆抗体。根据这些抗体和~(125)I 标记的hCG,hCGα,hCGβ和hLH 结合的情况,可将抗体分为三类。抗体BAH_2作用于hCG_α和β亚单位结合后所暴露出的抗原决定簇,抗体10E_5和8D_5特异地和hCGβ亚单位结合但不和hLH 结合。大多数抗体是作用于hCG 和hLHβ亚单位上共有的决定簇。实验显示这些单克隆抗体特异性优于多克隆抗体,它们亲合常数在5.3×10_7～7.2×10~(10)M~(-1)之间。     

两种单克隆抗体—hCG、hLH的制备及其临床应用

用半固体培养基筛选hCG和LH单克隆抗体。272株细胞克隆系分泌hCG抗体,hCGβ-4D6(亲和常数1.2×10~(10)m~(-1))和hCG_2β-_1EO(亲和常数8.7×10~(18)M)选作制备hCG免疫测定药箱。43株细胞克隆系分泌LH抗体,LH_(15)(亲和常数6.7×10~9m~(-1))和LH_(48α)(亲和常数5×10~8m~(-1))选作制备LH免疫测定药箱。设计放射免疫竞争测定法(RIA),固相免疫放射测定法(IMRA)固相免疫酶标测定法等三种方法测定hCG和LH浓度。hCG和LH免疫测定药箱灵敏度分别为3mIU/ml和2mIU/ml。月经规则妇女的LH峰可用一种30分钟的程序从尿样中测出。为尽可能减低激素之间的交叉反应,hCG免疫测定药箱系统加入LH_(130)单克隆抗体,LH免疫测定药箱系统加入hCGβ-4D6。实验结果表示hCG和LH水平可用不同免疫测定药箱测定之,其免疫交叉反应极低。     

人绒毛膜促性腺激素(hCG)免疫避孕研究近况

<正> 人绒毛膜促性腺激素(hCG)是胎盘绒毛膜合体滋养层细胞所分泌的一种糖蛋白激素,对早期妊娠的维持起重要的作用。从70年代初开始,人们为了研制避孕疫苗,对hCG、hCG-β亚单位(hCG-β)及其C 端多肽进行了广泛、大量的研究。本文简略介绍该研究的近况。一、hCG-β亚单位疫苗hCG 是由α和β两个亚单位组成,α亚单位与黄体生成素(hLH)、促甲状腺素(TSH)和卵泡刺激素(FSH)等激素的α亚单位相同,所以,用全hCG 分子免疫动物和人,就会与这几种激素发生交叉反应,导致严重的副作用;β亚单位在结构上与这些激素差异较大,因而具有一定的免疫特异性。Hearn 用hCG-β给狨猴主动免疫,能产生hCG 抗体,使早期妊娠的胚胎流产、中期妊娠胚胎吸收,对晚期妊娠则无作用;用其抗血清     

利用单克隆抗体研究人绒毛膜促性腺激素的结构与功能关系

利用一组抗人绒毛膜促性腺激素单克隆抗体研究它们与hCG及其亚单位结合以及在体内外中和hCG,hLH活性的能力。发现至少在hCG存在着6个抗原决定簇。其中与BAH_1,9C_2,10E_5,8B_5反应的决定簇和hCG生物活性有关,而与BAH_2和7F_3反应的决定簇与hCG生物活性无关。与抗体10E_5和8B_5反应的这两个抗原决定簇是hCGβ亚单位所特有的,它们不存在于hLH分子中。     

孕酮单克隆抗体的制备

本研究获得了三个分泌孕酮单克隆抗体的杂交瘤细胞株。这些高特异性抗体既和游离也和结合孕酮结合。这些抗体可和孕酮-牛血清白蛋白产生沉淀反应,但不沉淀牛血清白蛋白。它们可凝集覆盖有孕酮-牛血清白蛋白的羊红血球。它们的亲合力(Ka)在3～4.5×10~9M~(-1)之间。利用K(?)hlef 和Milstein 开创的杂交瘤技术制备激素单克隆抗体的工作开始不久,但已显示出巨大的潜力。利用这种技术获得大量均匀专一的孕酮抗体不仅有助于孕酮免疫测定的标准化,也将促进对孕酮在生殖过程中的作用的进一步了解。本文报道我们实验室利用杂交瘤技术获得的3个分泌孕酮单克隆抗体的细胞株。     

人绒毛膜促性腺激素的小鼠单克隆抗体(W14A)的制备及其特性

无论是足月顺产或是清除水泡状胎块之后,若能迅速识别出有发生绒毛膜癌危险的人群,对于成功的治疗极为重要。而系统检测血清中人绒毛膜促性腺激素(hCG)的变化则是早期诊断与治疗的关键之一。由于单克隆抗体具有对特定抗原的高度专一性,因此可用于临床的诊断与治疗。本文作者描述可与完整 hCG 及与 hCG-β亚单位反应的小鼠单克隆抗体 W14A 的制备及其性质。所用动物为 BALB/c 小鼠。首先以完整 hCG免疫小鼠(20μg,生物测试为3,000IU/mg),首次以 s·c·注射并加用完全 Freund 氏佐剂。然后于20和40天后加强免疫一次。最后于进行杂交技术前三     

雌二醇单克隆抗体的制备及性质

本文建立了5株稳定分泌抗雌二醇(E_2)单克隆抗体(McAb)的杂交瘤细胞株。其中1株McAb与雌酮、雌三醇交叉反应率分别为8.1%和0.5%,与其它7种甾体化合物交叉反应率均<0.01%。抗体亲和力为1.8×10~(10)L/M,腹水抗体滴度:1:400,000。其所做的剂量反应曲线稳定,灵敏度1pg,样品回收率:104±15.8%(n=7),批内变异系数:4.07%(n=12),批间变异系数:8.82%(n=6)。该株McAb可望取代常规抗血清应用于雌二醇放射免疫测定等方面。     

重组人绒毛膜促性腺激素β-亚基对C57BL小鼠免疫应答反应及抗血清特征的研究

采用福氏性剂，对C57BL小鼠进行重组hCGβ和天然hCGβ免疫应签反应的比较研究，结果表明：两者引发的抗血清性质相似，与hCG有较高的亲和力（K（γβ）≈5．86×108／mol／L，Kaβ≈8．18×108／mol／L）；抗血清能有效地抑制（125）I－hCG与睾丸受体的结合，结果提示重组hCGβ抗血清与天然hCGβ抗血清相似，具有中和hCG的生物活性能力，重组hCGβ可作为免疫避孕疫苗的抗原。     

AF-113的抗生育作用研究

大鼠,小鼠和仓鼠受孕后第1(d_1),第2(d_2)和第7(d_7)天灌胃(Po)AF-113 0.56—15mg/kg 有明显的抗生育作用。其抗着床ED_(50)大鼠和小鼠分别为1.03(0.66—1.62)和0.54(0.14—2.12)mg/kg,仓鼠为10.0(7.3—13.8)mg/kg。AF—113使幼年大鼠子宫增重的效价为EE 的1.9%;能抑制大鼠子宫内膜蜕膜瘤生长,抑制幼兔子宫内膜增生,但本身无孕激素活性;体外能与大鼠、小鼠和仓鼠子宫胞浆雌激素受体结合,其抑制~3[H]-雌二醇与雌激素受体相结合的IC_(50)分别为2.5±1.1×10~(-5),4.3±2.6×10~(-6)和5.0±0.3×10~(-5)M     

溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响

目的:探索与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响。方法:提取溶脲脲原体总DNA,用自行设计的引物扩增UreG全序列,PCR产物经TA克隆后,用BamHⅠ和HindⅢ双酶切,克隆到表达载体pET-28a(+)中。在E.coliBL21中,异丙基-β-硫代半乳糖苷(IPTG)诱导下表达His融合蛋白。用亲和层析纯化的重组蛋白免疫BALB/c小鼠,获取多克隆抗体。观察该抗体对小鼠体外受精的影响。结果:对表达重组蛋白的质粒进行DNA测序以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA检测证实,免疫小鼠产生了高效价的抗体。该抗体能够抑制小鼠精卵的融合。结论:与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体,可导致小鼠精卵结合率降低。     

Comparison of different regimens of human gonadotropins for superovulation of rhesus monkeys: Ovulatory response and subsequent luteal function

This study was designed to identify suitable treatment regimens of human gonadotropin for superovulation of rhesus monkeys. At menses, female monkeys were given one of three regimens: Plan A [days 1 to 6, 60 IU human follicle-stimulating hormone (hFSH); days 7 to 9, 60 IU hFSH/60 IU human luteinizing hormone (hLH)], Plan B [days 1 to 3, 75 IU FSH/20 IU LH; days 4 to 6, 60 IU FSH/20 IU LH; days 7 to 9, 45 IU FSH/45 IU LH], or Plan C [days 1 to 9, 60 IU FSH/60 IU LH]. On day 10, human chorionic gonadotropin (hCG; 1000 IU) was administered. Serum estrogen levels peaked on the day of hCG treatment (day 10) in Plans A and C but earlier (day 8) in Plan B. An oviduct lavage recovered 1 to 3 oocytes in Plan B but 3 to 13 oocytes in the other treatment groups. Peak progesterone levels in the luteal phase were greater (P<0.05) in animals receiving Plan A or C than Plan B. Regardless of treatment group, progesterone levels declined abruptly 7 days after ovulation induction; the length of the luteal phase in all groups was significantly less than that of normal menstrual cycles. We conclude that regimens of hFSH and hLH (i.e., Plans A and C), followed by hCG, reliably superovulate rhesus monkeys. However, the premature decline in luteal function around the typical time of implantation may compromise pregnancy initiation and maintenance.     

Monoclonal antibodies to human chorionic gonadotropin and its beta-subunit

Using the technique of somatic cell fusion, monoclonal antibodies to human chorionic gonadotropin (hCG) and its beta-subunit were produced. Spleen cells from immunized mice were fused with the myeloma line NS-1 using 50% polyethylene glycol and cultured in a selection medium. A sensitive enzyme immunoassay was performed for antibody screening using immobilized solid-phase antigen and anti-mouse IgG Fab' (rabbit)-beta-D-galactosidase complex. Three double-cloned hybridomas (named 4H10, 4G2 and 1D12) were obtained. Anti-hCG 4H10 antibody reacted with hCG, the alpha-subunit of hCG (alpha-hCG) and human luteinizing hormone (hLH); and anti-hCG 4G2 antibody reacted with hCG and the beta-subunit of hCG (beta-hCG). It was interesting that anti-beta-hCG 1D12 antibody had the capacity to bind with free beta-hCG but not with intact hCG, which suggests that the 1D12 antibody can recognize a determinant site that is unique to beta-hCG. This unique epitope in beta-hCG might be expressed in or near a part which is hidden in the intact hCG molecule which is composed of alpha- and beta-subunits.     

Characterization of anti oLH beta-antibodies acting as contraceptives in rhesus monkeys. I. In vitro binding properties

Female monkeys actively immunized with oLHbeta are infertile when circulation antibody titers become sufficiently high to bind 30% of an iodinated hCG standard. We report here the extensive characterization of the oLHbeta antibodies. Their binding affinity and capacity for human and rhesus gonadotropins were determined. The affinity was high (Ka - 10(9) M-1) and was similar for high, medium and low titer antisera. In contrast, the binding capacity for hCG correlated well with antibody titers (n = 18, r = 0.888, P less than 0.001) and ranged from 0.09 to 8.3 x 10(-7)M. Column chromatographic analysis showed that anti-oLHbeta was mostly IgG. A temporal increase of affinity ('maturation') after booster was absent in the majority of monkeys. Cross-reaction between hCG and hLH as well as rhCG and rhLH was high. The latter, however, did not interfere with regular cycles and ovulation, suggesting that oLHbeta may be a useful antigen for human contraception.     

Luteal function following ovarian stimulation in rhesus monkeys for in vitro fertilization: atypical response to human chorionic gonadotropin treatment simulating early pregnancy

This study determined if corpora lutea of hyperstimulated cycles in rhesus monkeys could be "rescued" by the pregnancy signal, chorionic gonadotropin (CG), given at the typical time of implantation. At menses, female monkeys received human follicle-stimulating hormone (hFSH, 60 IU, days 1 to 6) followed by human menopausal gonadotropin (hMG, 60 IU hFSH/60 IU luteinizing hormone [hLH], days 7 to 9). On day 10, human chorionic gonadotropin (hCG) was given to mimic the LH surge. Nine days later, a regimen of daily increasing doses of hCG (15 to 360 IU twice a day) was initiated to simulate rescue of the corpus luteum in early pregnancy. Serum levels of progesterone (P) increased through day 5 of the luteal phase but then declined. Circulating levels of bioactive LH were significantly less on days 7 to 9 of the luteal phase than at this stage in the natural cycle. The hCG regimen extended (P less than 0.05) the luteal phase in five of six animals. The hCG treatment elicited a persistent increase (P less than 0.05) in circulating P levels, rather than a transient rise typical of normal or simulated pregnancy in natural cycles. The authors conclude that (1) corpora lutea of hyperstimulated cycles can respond to CG, but (2) there are differences in luteal function during both the luteal phase and simulated early pregnancy that may be due to inadequate luteal development or the abnormal gonadotropin milieu existing after ovulation or both.     

An improved immunogen for anti-human chorionic gonadotropin vaccine eliciting antibodies reactive with a conformation native to the hormone without cross-reaction with human follicle stimulating hormone and human thyroid stimulating hormone

Beta-subunit of human chorionic gonadotropin (hCG) was associated with alpha-subunit of ovine luteinizing hormone (OLH) to create a heterospecies dimer (HSD) which has a higher steroidogenic potency than the homologous dimer of alpha hCG and beta hCG in the mouse Leydig cell bioassay. The properties and merits of the antibodies induced by this HSD and beta hCG linked to carrier(s) were investigated in rodents and in a subhuman primate species. The antisera had, in both cases, high affinity for binding with hCG (K alpha = 10(9)-10(10) M-1). The mean (+/- S.E.M.) bioneutralization capacity as a percentage of immunoreactivity (determined by radioimmunoassay (RIA)) of the antibodies generated by the HSD-carrier in rats and bonnet monkeys was higher in comparison with those induced by beta hCG linked to carrier (80 +/- 2.3% vs. 63 +/- 1.5% in rats, and 65 +/- 1.9% for HSD vs. 44 +/- 3.7% in monkeys). None of the sera gave any evidence of cross-reactivity with human follicle stimulating hormone (hFSH) and human thyroid stimulating hormone (hTSH).