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。     

人绒毛膜促性腺激素β亚单位

人糖蛋白激素如黄体生成素(hLH),促滤泡激素(hFSH),促甲状腺激素(hTSH),以及人绒毛膜促性腺激素(hCG)都由两个亚单位组成。各激素的α亚单位的氨基酸顺序几乎完全相同,而β亚单位的结构则均异,故各激素的生物活性都由其β亚单位决定。 hCG的分子量为39,000。α亚单位(α-hCG)为16,000,有92个氨基酸;β亚单位(β-hCG)分子量为23,000,有145个氨基酸,其中97个和hLH的β亚单(β-hLH)相同,因此hCG和hLH具有类似生物活性。     

hCG单克隆抗体特性的研究

用hCG 免疫BALB/C 小鼠,取其脾细胞和NS1骨髓瘤细胞融合,得到二株能分泌识别hCG 和hCG-β亚单位的单克隆抗体的杂交细胞株。其抗体A_(46)和A_(24)对hCG 的亲和力用Scatchard 法测得平衡常数分别为1.60×10~9M~(-1)和2.33×10~9M~(-1),对hCG-β的平衡常数为0.62×10~9M~(-1)和2.50×10~9M~(-1)。A_(46)抗体还能识别hLH,和hCG-α、hFSH 和hTSH 的交叉反应很小。而A_(24)抗体和hLH 只有2%的交叉反应,和其它几种激素几乎无交叉反应。这两种单克隆抗体都能阻断hCG 在雄小鼠体内刺激睾酮产生的作用。实验结果还提示A_(46)和A_(24)抗体在hCG 分子上所识别的抗原决定簇很接近,并可能都和hCG 分子的生物学活性的表达有关。     

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     

β-hCG酶联免疫目测法检测已孕和非孕闭经250例观察

人绒毛膜促性腺激素(hCG)是由滋养层合体细胞分泌的一种糖蛋白,分子量约4,700,具有蛋白分子的四级结构,由一蛋白核心及α、β糖分子侧链组成,侧链尾端常为涎酸,与hCG的生物活性有关。hCG的α侧链与hFSH及hLH的α侧链结构相似,三者具有免疫交叉反应及类似的生物效应,而hCG的β侧链则具有免疫及生物学特异性。本文所述的β-hCG酶联免疫目力测定与放射免疫测定,均建立在β亚单位的结构基础上,是一敏感性较高的可用以诊断早孕及滋养叶细胞疾病的方法。 hCG为水溶性蛋白,在孕卵着床后即由滋养叶合体细胞产生,孕8～10周血浓度达高峰,测试体液(血、尿、脑脊液)中hCG有助于对早孕及滋养叶细胞疾病     

抗hCG单克隆抗体作妊娠试验试剂

免疫学妊娠诊断由于简便已广泛用于临床。已知hCG和hLH有类似的氨基酸结构,抗hCG抗体与hLH之间必然有免疫交叉性,特别是测定低浓度hCG时由于hLH的交叉,可能出现错误的结论。由于细胞工程学的发展,单克隆抗体的应用已成为可能。     

绒毛膜促性腺激素(hCG)进展

绒毛膜促性腺激素(hCG)是一种糖蛋白,由胎盘产生,在孕妇血与尿中都大量存在,尤其在早期妊娠阶段,故能用生物法或免疫法作测定以诊断早孕。在非妊娠期,hCG出现于血或尿中为病理现象,指示有肿瘤组织分泌此激素。hCG的生物活性同hLH有类似处,但不完全相同。一、分泌部位:过去认为hCG由细胞滋养层细胞分泌,但Midgley等用荧光免疫法发现抗hCG血清定位于合体滋养层细胞内、电镜检查发现合体细胞内有发育良好的内质网。细胞免疫化学法指出合体细胞糙面内质网的内质网池内能找到hCG。故现一致认为hCG主要由合体细胞分泌。     

单抗技术在分析和研究hCG抗原决定簇中的应用

<正> 绒毛膜促性腺激素(hCG)是由二条多肽链亚基聚合而成的糖蛋白激素,其α-亚基结构是与垂体前叶分泌的糖蛋白激素类(hFSH,hTSH,hLH)相同,来源于同一基因,而其β-亚基是特异的,受七个基因控制。尽管 hCG 的氨基酸序列已经明确,而其立体构象、生物活性决定部位、亚基之间的聚合部位及激素和受体的作用部位仍在探索中,作者就从事上述专题研究中所阅文献予以综述。     

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。     

尿β-核心hCG:对孕早期(11～14周妊娠)非整倍体的筛选

在孕中期对Down氏综合征的生化筛选最有诊断价值的标记是人绒毛促性腺激素(hCG)或其游离β-亚单位。该激素的完整分子是含一个特殊β-亚单位与α-亚单位共价结合的二聚体。在Down氏综合征孕妇血浆中完整hCG和游离β-亚单位水平升高,在β-亚单位残基47和48或44和45之间存在切     

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.     

Gonadotropin receptors of human corpus luteum during menstrual cycle and pregnancy.

Specific high-affinity low-capacity binding of human chorionic gonadotropin (hCG) and lutropin (hLH) was demonstrated in the plasma membrane fractions of human corpora lutea. The number of binding sites for both hormones increased from early to late luteal phase, whereas regressing corpus luteum from proliferative phase did not bind either hormone. On the basis of apparent dissociation constants the affinity of the receptor for hLH is highest during early luteal phase and decreases toward the end of the cycle, which may reflect an increasing insensitivity of the corpus luteum to circulating hLH. By contrast, the affinity of the receptor for hCG is highest in the midluteal phase. There are gonadotropin binding sites in the human corpus luteum also during pregnancy, but they are saturated by endogenous hCG. Evidence for this was obtained by elution of hCG with 0.15M sodium chloride at pH 2.3 from washed plasma membrane fractions of luteal tissue from six to 16 week's gestation. After acid treatment and neutralization these preparations showed specific binding for 125I-labeled hCG, but not for 125I-labeled hLH. Our results demonstrate a shift in the balance of affinity of the gonadotropin receptor from hLH to hCG during the course of luteal phase, and during pregnancy the binding sites appear to be available for hCG only.     

Expression of a recombinant bifunctional protein from a chimera of human lutropin receptor and human chorionic gonadotropin beta-subunit

Human lutropin (hLH) and human chorionic gonadotropin (hCG) are structurally and functionally similar and play important roles in reproduction via a common gonadal receptor (LH-R). However, hormone specific hCG-beta subunit contains 24 additional amino acid carboxyl terminal peptide (CTP), which produce specific antibodies to hCG-beta with little cross-reaction with LH. A chimeric protein containing both hLH-R and hCG-beta would provide a unique bifunctional antigen for immunocontraception. In this study is described the synthesis of a chimeric DNA construct of full-length of hLH-receptor and hCG-beta and its expression in Sf9 cells to produce a bifunctional protein. Recombinant protein was recognized by antibodies to LH-R as well as anti-hCG-beta in Western Blots, thus indicating the preservation of immunological epitopes for both hLH-R and hCG-beta in the chimera. Specific ligand binding of recombinant hLH-R component was demonstrated by the displacement of bound labeled hCG at increasing concentrations of unlabeled hCG, indicating that, the presence of hCG-beta component of the chimera did not interfere with the binding of hCG to LH-R. hCG-beta was also present in the recombinant chimeric protein as shown by a specific hCG-beta chemiluminescence assay. Treatment of transfected Sf9 cells with hCG induced dose-dependent increase in the stimulation of intracellular cAMP production, which showed that the ligand binding had functional activity. These results demonstrate that the chimeric DNA construct of hLHR-hCG-beta expressed a bifunctional protein containing both hLH-R and hCG-beta activities, which could provide a unique potential antigen for immunocontraception in vertebrates.     

Immunobiological properties of a carboxy-terminal 53-amino acid peptide of the beta subunit of human chorionic gonadotropin

Lengthening of the carboxy terminus unique region of beta-hCG from 30 to 45 amino acids was found in previous studies to improve immunogenicity and hormone neutralization capacity. The present study was carried out to determine whether further elongation of the peptide to 53 amino acids enhances to hormone neutralization capacity without loss of specificity characteristics. The peptide 93--145 of beta-hCG with substitution of cysteines at 93, 100 and 110 by alpha-aminobutyric acid was synthesized by solid phase and conjugated to tetanus toxoid by an active ester method. Rabbit antibodies against this conjugate reacted with CTP-53 and CTP-45 with a parallel slope in anti-CTP-53-[125I]Tyr-CTP--53 radioimmunoassay system. Other CTPs, e.g. CTP-26, CTP-31 and CTP-35 competed with lower efficiency; 50% inhibition of binding was obtained with 10-100 pmol/tube with these peptides instead of 0.5 pmol/tube for the homologous CTP-53. Anti-CTP-53 reacted with both beta-hCG and hCG but around twenty times greater amount of hCG was required to give 50% inhibition of binding as compared to CTP-53. The antigen binding capacity of anti-CTP-53 was around 4000 ng/ml for CTP-53 and 25 ng/ml for hCG. The anti-CTP-53 sera retained non-cross-reactivity with hLH as determined by direct binding with [125I]hLH and by competitive inhibition of CTP-53 binding with anti-CTP-53. Anti-CTP-53 neutralized the bioactivity of hCG in the Leydig cell bioassay and in the mouse uterine weight gain assay. Anti-CTP-53 antibodies were about three times more effective than anti-CTP-45 in their capacity to neutralize the bioactivity of hCG, though still substantially poorer than anti-beta-hCG sera in this respect.     

Reverse passive hemagglutination assay for human chorionic gonadotropin in urine using monoclonal antibody

Aiming to find a urinary hCG immuno-assay which is specific, sensitive and easy to perform, a reverse passive hemagglutination reaction was studied by using sheep red blood cells (SRBC) coupled with monoclonal antibodies (Mab) to hCG. Three Mabs (5D4, 6E4, 2F8) with different specialty were used for the study. Mab 5D4 reacted to hCG, hCG-beta, and LH but not to hCG-alpha. Mab 6E4 reacted to hCG, hCG-alpha and LH, but not to hCG-beta. Mab 2F8 reacted to hCG but not to hCG-alpha, hCG-beta, or LH. All three Mabs were IgG1. SRBC were treated with glutaraldehyde and then with tannic acid. These treated SRBC were coupled with IgG(2mg/ml) of each anti hCG-Mab. For assays, 30 microliters of 1:1 mixtures of two different Mab-coupled SRBC and 30 microliters of standard hCG or urine samples were mixed in wells of microtiter plates and reacted for 60 min at room temperature. Among three different combinations, the couple 5D4-SRBC and 2F8-SRBC were most sensitive and specific for hCG assays and the minimum amount of hCG and LH detected in this combination assays was 12.5 mIU/ml and 800 mIU/ml, respectively. Some clinical data obtained by applying this assay were presented.     

Characteristics of antibodies raised to carboxy-terminal peptides of hCG beta subunit

Natural and synthetic peptides representing COOH terminal sequences of the beta subunit of human chorionic gonadotropin (hCG) were coupled to tetanus toxoid and rabbits immunized with the conjugates. Sera were evaluated for antibody levels, antibody affinity and antibody class. Conditions for appropriate estimation of these parameters were also studied.Findings revealed that iodination of hCG or peptides did not alter their immunological properties and that reaction of labelled antigens with antisera require differing periods of time for the establishment of equilibrium. Peak titers to the hCG antigens were reached at 70 days of immunization. Antibodies to natural peptide 109–145 of β-hCG and synthetic peptides 109–145, 111–145, and 115–145 reacted to hCG approximately 90% as well as they reacted to the peptide used for immunizations whereas antibodies to synthetic peptide 125–145 reacted only 60–70% as well. Mean antibody affinities to peptides were not significantly higher than affinities of the same sera to hCG. The affinity of antisera to synthetic peptide 125–145 to both antigens and the mean ratio of hCG affinity : peptide affinity was somewhat lower than those to longer peptides. After 70 days of immunization, antibodies were predominantly of the IgG class.The findings indicate that antibodies raised to natural or synthetic peptides of the COOH region of hCG beta subunit are highly cross-reactive to native hCG but have affinities somewhat lower than those generated to the intact hormone or its beta subunit.     

The use of a radioimmunoassay specific for human chorionic gonadotropin in patients with molar pregnancy and gestational trophoblastic disease

Serum human chorionic gonadotropin (hCG) activity is compared in 14 patients under treatment for gestational trophoblastic disease (GTD) using both a radioimmunoassay (RIA) specific for hCG and a nonspecific rapid solid-phase RIA which measures both luteinizing hormone (hLH) and hCG. The results indicate that the nonspecific RIA is adequate for the diagnosis and management of patients with GTD when the hCG titer is above endogenous hLH levels, but a specific RIA is required to ensure complete remission and to detect early recurrence during follow-up.