重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

ELISA检测抗透明带抗体方法的建立及初步临床应用

透明带 (zonapellucida ,zp)抗原是哺乳动物卵母细胞分泌并被覆于卵细胞外的一层基质 ,属糖蛋白[1 ] 。zp抗原具有很强免疫原性 ,通过动物实验证实 ,透明带抗体 (Azp)能阻遏精子吸附和穿透zp ,阻碍囊胚着床前zp的生理性剥脱 ,以干扰着床 ,产生抗生育作用 ,另外 ,Azp还能影响卵子的发育及生长[2 ] 。相当部分不明原因不孕症妇女通过对抗透明带抗体 (Azp)检测 ,而找到了不孕的原因。故Azp诊断对不孕症原因的判断及治疗有很大意义。材料和方法一、对象 :不孕症妇女 6 5例 ,年龄 2 5～ 39岁 ,婚后性生活正常 ,≥ 2年未孕 ,经子宫、输卵管造影 …     

人抗卵透明带抗体阳笥血清对小鼠体外受精和早期胚发育的影响

本研究在小鼠体外受精和早期胚胎发育中添加抗卵透明带抗体阳性不孕妇女血清(n＝11）,结果显示：与对照组相比较,6例完全阻断受精和发育（P＜0.01）;2例仅阻断受精,不明显抑制发育;3例则与对照组无显著性差异（P＞0.05）。提示人抗卵透明带抗体的主要效应是阻断受精,并可以进一步抑制胚胎早期发育,但效应的程度表现出个体差异。     

不同人群血清SARS冠状病毒抗体检测及其意义

目的　通过对不同人群SARS冠状病毒IgG抗体 (SARSCoVIgG)检测 ,明确该抗体对SARS的诊断意义。方法　采用酶联免疫法 (EIA)、间接荧光法 (IFA)和免疫印迹法 (WB)检测抗 SARSCoVIgG。结果　对 117例临床确诊为SARS患者的 336份系列血清检测表明 ,SARS病人血清抗 SARSCoVIgG最早于发病后第 9天阳转 ,其阳性率随病程延长而上升 ,于发病后 5～ 9、10～ 14、15～ 19、2 0～2 4和 2 5d以上抗 SARSCoVIgG阳性率分别为 12 .5 % (1/8)、73.9% (17/2 3)、91.5 % (43/47)、96 .6 %(5 7/5 9)和 10 0 % (198/198)。应用EIA初筛 12 2 3名非SARS人群 (包括 36 7名在SARS病房工作 1个月以上的医务人员 ,4 3名在生活中与临床确诊的SARS病人有密切接触史者 ,以及 813例未暴露于SARSCoV人群 ) ,其中 2 8名为抗 SARSCoVIgG弱阳性 (A <0 .5 ) ,但用 2种IFA和WB检测均为阴性 ,说明EIA初筛为假阳性。结论　应用EIA检测抗SARSCoVIgG有助于中晚期SARS病人的诊断。对EIA初筛为抗 SARSCoVIgG弱阳性的标本 (A <0 .5 ) ,应用其他方法如IFA和WB检测 ,以排除假阳性。     

北京流感流行前后人群血清抗体水平检测分析

近几年我们常年进行人群流感抗体监测 ,北京1998年 12月开始流感暴发流行 ,现将暴发流行前后的血清抗体结果比较分析报告如下。1　材料与方法1 1　抗体检测用的流感毒株代表株　共选用 8株代表株 :甲 3型流感毒株 (Ｈ3Ｎ2 ) 2株 ,甲 1型(Ｈ1Ｎ1) 4株 ,乙型 2株。1 2　抗体检测用的霍乱弧菌液　霍乱弧菌液由国家流感中心提供。1 3　三类血清标本来源　流行前血清标本 :1998年11月随机采集北京卫戍区 5112 5部队战士静脉血10 5份 ,分离血清 ,- 30℃保存待测。流行后血清标本 :1999年元月 15日随机再次采集 5112 5部队战士静脉血 98份 ,分离血…     

不同人群中SARS-CoV-2抗体检测的作用价值

目的:探讨新型冠状病毒（severe acute respiratory syndrome coronavirus 2,SARS-CoV-2）特异性IgM和IgG抗体检测应用于不同人群新型冠状病毒肺炎（corona virus disease 2019,COVID-19）诊断和筛查的作用价值。方法:回顾性分析2020年...     

对不育不孕者抗精子抗体和抗子宫内膜抗体的检测分析

不育(孕)是生殖医学研究的一个热点,在多种导致不育(孕)因素中,相当一部分为免疫不育,为协助免疫不育的临床诊断,我们对43例不育(孕)症病人做了抗精子抗体和抗子宫内膜抗体的检测,观察结果如下.     

反复流产和不孕不育症血清中抗-HCG抗体检测分析

反复流产（RSA）的病因十分复杂，除染色体异常、内分泌失调、生殖结构畸形和感染以外，均有60％～70％为原因不明。近年来研究认为免疫因素是一类重要原因。不孕症是妇科常见疾病，原（继）发不孕也存在免疫学因素，本文旨在检测复发流产和不孕症患者血清中抗-HCG抗体（AH—CG），以了解其之间关系。     

抗卵透明带免疫应答中IL-2和IL-4含量的变化

为了观察卵透明带抗独特型抗体免疫抗生育中ＩＬ ２、ＩＬ ４两种细胞因子含量的变化,分别用猪卵透明带（ＰＺＰ）抗原、鼠抗ＰＺＰ单克隆抗体（１７Ｄ３ｍＡｂ）亲和提纯的透明带抗原和ＰＺＰ的抗独特型抗体（Ａｂ２）免疫小鼠,通过脾细胞体外诱生,测上清液中的ＩＬ ２和ＩＬ ４。结果发现Ａｂ２免疫组主要介导的是Ｔｈ２型免疫反应,而ＰＺＰ免疫组和１７Ｄ３ｍＡｂ靶抗原免疫组ＩＬ ２水平明显升高,可能介导的是Ｔｈ１型免疫反应。结果提示Ａｂ２所模拟的抗原决定簇在ＺＰ免疫避孕的研究中是有前途的。     

Anti-ZP3 Antibodies Binding to the Human Zona Pellucida: Effect of Oocyte-Storage Conditions

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions. METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm-ZP binding capacity under different HZA conditions. RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4°C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt-stored, or frozen with 1,2 propanediol. CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol-treatment while the glycoprotein-receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.     

Does In Vitro Fertilization (IVF) Influence the Levels of Sperm and Zona Pellucida (ZP) Antibodies in Infertile Women?

The levels of sperm and zona pellucida antibodies in 250 women divided into four groups according to number of recurrent IVF failures (1–4) were analysed and compared with results of a control group of 211 unexplained infertile women never treated by IVF. Sperm antibodies in serum and in ovulatory cervical mucus were determined by mixed antiglobulin reaction (MAR) test, serum zona pellucida antibodies were detected using passive haemagglutination and ELISA. These tests showed increased occurrence of zona pellucida antibodies in women after repeated IVF. Zona pellucida antibodies were found in 20% after one unsuccessful IVF (similarly to 27% in the control group), but in 64% after two, in 91% after three and in 4 of 5 cases after four IVF failures. Sperm IgG, A, M and E antibodies in serum and in ovulatory cervical mucus do not seem to be influenced by IVF procedure. The results show evolution of autoimmune process due to repeated ovarial intervention during oocyte collections. Presence of zona pellucida antibodies, on the other hand, may become a cause of IVF failure.     

Zona Pellucida Antibodies in a Group of Women With Idiopathic Infertility*

ABSTRACT: The presence of zona pellucida antibodies was sought in a group of ten women with infertility of unknown cause. The reaction of these sera with ova was compared to the reaction obtained with a control antizona pellucida serum produced by the immunization of a rabbit with 8,000 solubilized hamster zonae pellucidae. This antiserum reached a maximum titer of 1 in 256 as measured by an immunoprecipitation assay and of 1 in 1,024 as measured by indirect immunofluorescence. It is tissue-specific but is not species-specific. With the indirect immunofluorescence technique on ova obtained from different species, the sera of eight of the ten patients gave a positive reaction with at least one type of test ova: three patients gave a positive reaction with hamster ova, six with porcine ova, and two with monkey ova. One infertile woman presented a positive reaction on hamster ova and another on pig ova as detected with all three types of γ-globulins. Two other patients presented a positive reaction with the anti-IgG and -IgM on porcine ova. The titer of the reaction never rose above one in four in any of the positive cases. Adsorption of the sera with fresh erythrocytes from human, hamster, and porcine blood did not change the titer or the intensity of the fluorescent reaction on ova. This indicates that the positive reactions are not due to nonspecific heteroagglutinins. Adsorption of the sera with formalin-fixed erythrocytes from human and porcine blood completely abolished the fluorescent reaction. However, the latter treatment, applied to a dihydrotestosterone antiserum and a β-hCG antiserum also greatly diminished the binding of hormones to these antisera; fresh erythrocytes did not adsorb the antibodies in these two antisera. This demonstrates that formalin-fixed erythrocytes are not suited for adsorption of sera before testing for specific autoantibodies to the zona pellucida in infertile women. None of the sera from the infertile women gave a positive reaction with human pre-ovulatory or post-ovulatory ova or with frozen sections of human ovary. None of the sera produced an inhibition of in vitro fertilization of hamster ova. On the contrary, the control zona pellucida antiserum completely blocked in vitro fertilization. Furthermore, in a group of pregnant women, 2 of 11 exhibited a positive reaction with hamster ova while 6 of 28 showed a positive reaction with porcine ova. These results suggest the presence of low titer zona pellucida antibodies in 8 of the 10 infertile women. The importance of these antibodies in their infertility remains to be established.     

Identification of Epitopes of Monoclonal Antibodies to Porcine Zona Pellucida 3β Glycoprotein,a Homologue of the Mouse/Human Sperm Receptor

PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3β, a homologue of the designated primary sperm receptor in mice and humans. METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3β-deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme-linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3β in a competitive inhibition ELISA was investigated to confirm the above findings. RESULTS: Reactivity of the mAbs with the pin-bound peptides in ELISA-identified epitopes for mAb-451 to EEKLVF (166–171) and mAb-462/470 to FKAPRP (250–255) amino acid residues. mAb-30 recognized QPVWQDEGQRLR (23–34) and VICRCC (316–321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23–34 and 316–321 for binding of mAb-30 to pZP3β revealed the epitopic domain to be 23–34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb-30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue. CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3β or its homologues in other species.     

Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

ABSTRACT: In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium-3,5-diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona-IgG was raised by immunization of female rabbits with 500 heat-solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (M_r = 42,000), ZP II/1 (M_r = 67,000), ZP II/2 (M_r = 32,000), ZP III/1 (M_r = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high-pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O-glycosidic-glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.     

Characterization of Antibodies Generated Against a Conserved Portion of Oviductal Glycoprotein (OGP) and Endogenous Hamster OGP and Their Ability to Decrease Sperm Binding to the Zona Pellucida In Vitro

PROBLEM: The effect of antibodies generated against hamster oviductal glycoprotein (OGP) on sperm binding to the zona pellucida (ZP) was evaluated. METHOD OF STUDY: Antibodies against a 17-amino-acid sequence of the OGP core protein (amino acids 52–68) and the denatured hamster OGP protein were generated, characterized, and tested in an in vitro sperm binding assay. RESULTS: Sperm binding was significantly decreased (P < 0.05) when oviductal oocytes were incubated for 2 hr with 4 or 8 mg/ml of immune IgG of both antibodies when compared with normal rabbit IgG. A fluorescence assay showed binding of both antibodies to the endogenous OGP associated with the ZP of ovulated hamster oocytes. CONCLUSIONS: These results suggest that OGP may be a potential immunocontraceptive target because both antibodies significantly decreased sperm binding to the ZP of oviductal oocytes. Immunocontraception may be accomplished by attempting to generate active immunity to a recombinant OGP, to the region selected in this study (amino acids 52–68) or to some other region of the core protein.     

Occurrence of the Interference of Sperm-Associated Antibodies on Sperm Fertilizing Ability as Evaluated by the Sperm-Zona Pellucida Binding Test and by the TEST-Yolk Buffer Enhanced Sperm Penetration Assay

PROBLEM: This study was performed to evaluate the occurrence as well as the level of the interference of sperm-associated antibodies on fertilization process. METHOD: Motile sperm suspensions from 28 infertile patients with high degree of autoimmunization against the sperm head were tested with the zona pellucida (ZP) binding test and with the sperm penetration assay (SPA) enhanced with TEST-yolk buffer. Both tests were also performed using donor sperm exposed and non-exposed to the patients' circulating sperm antibodies. RESULTS: A low ZP-binding was exhibited by sperm from 50% of patients with normal semen profile. All normozoospermic patients with low ZP-binding showed circulating sperm-antibodies with inhibitory effect on ZP-binding, while no patient with normal ZP-binding showed circulating sperm-antibodies with inhibitory effect. No normozoospermic patient exhibited a negative SPA result, and only in 16% of cases the penetration index was slightly less than 2 (the lowest value exhibited by fertile controls). Circulating antisperm-antibodies did not significantly affect the hamster egg penetration. CONCLUSION: Even in the presence of high degree of autoimmunization against the sperm-head, sperm fusion with oolemma is not impaired after sperm preincubation with TEST-yolk buffer, while an impairment of the ZP-binding is demonstrable in half cases, when non-immunologic factors are excluded. A substantial role in this interference is likely exerted by IgG antibodies transuded from the blood into the genital tract.