| 题名 | 抗顶体反应人精子单克隆抗体的制备和鉴定 |
| 作者 | 王云美 |
| 学位类别 | 硕士 |
| 答辩日期 | 1991 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 贲昆龙 |
| 关键词 | 人精子 获能 顶体反应 单克隆抗体 精子抗原 |
| 中文摘要 | 精子从男性生殖道释放后必须经过获能和顶体反应，才能与卵子结合。获能和顶体反应后，精子细胞内和精子表面的许多大分子都会发生改变。利用这些变化，可探索新的避孕方法和男性不育诊断及治疗的新途径。本文将正常人精子于体外在BWW－BSA培养基中获能，用钙离子载体A23187诱导人精子进行顶体反应，以三染和金霉素荧光染色两种方法检测这些精子的顶体反应率为50％左右，然后用这些经顶体反应处理的新鲜人精子（AR sperm）作为抗复，腹腔免疫Balb/C小鼠，利用最近发展起来的半固体培养及普通的液体培养方法制备单抗。分别用未经任何处理的人精子（NT sperm）及上述顶体反应的人精子包被酶标板，用ELISA法检测所得杂交瘤细胞分泌的抗体，得到了近百株阳性杂交瘤，其中已克隆并得到腹水的有23株，并对这些单抗进行了以下鉴定：腹水滴度测定；抗体分类；与人白细胞系U937，Raji和Jurkat的交叉反应；在NT和AR人精子、树（左鼠右句）和小鼠精子上的荧光定位；单抗对人精子的凝集（SA）和制动（SI）试验；免疫印迹测定单抗相应抗原的分子量。主要结果如下：1.单抗与NT和AR精子反应的ELISA结果表明，有12个单抗主要与AR精子抗原发生反应（A组抗体），6个单抗主要与NT精子抗原反应（B组抗体），而另外5个则与这两种精子抗原都呈阳性反应（C组抗体）。2.在得到的23个单抗中，绝大多数（21个）为IgM，只有两个分别是IgC_1和IgG_(2a)。3.23个单抗与白细胞系的交叉反应强度不同。A组单抗的交叉反应有的较强，有的较弱，有的居中；B组单抗的交叉反应为弱阳性或阴性；C组单抗则呈现要么很强、要么很弱的交叉反应。4.所得单抗的荧光定位主要在赤道板和中段，未发现定位于顶体及顶体后的单抗，而国内外其他实验室已获得的单抗，主要定位在顶体。某些单抗在两种不同精子（NT sperm, AR sperm）上有不同的荧光定位。这些结果表明，AR精子的免疫原性是十分特殊的，它明显不同于NT精子。5.有9个单抗显示较强的精子凝集作用，另有9个单抗的凝集作用稍弱，未发现有精子制动效应的单抗。6.免疫印迹结果表明，有9个单抗的靶抗原是蛋白质类物质，其分子量为16－146kDa，其余14个单抗的免疫印迹呈阴性反应。有关这些单抗及其抗原的鉴定仍在进行中。其中10个单抗已送世界卫生组织（WHO），参与WHO的抗人精子抗原的单克隆抗体的研究计划。 |
| 英文摘要 | Spermatozoa released from the male genital tract have to undergo capacitation and acrosome reaction(AR) before they are capable of binding to the oocyte. Changes of sperm components have been found after capacitation and AR. Based on investigating these changes, the new contraceptive methods and the treatment of the male infertility might be approached. In the present study, fresh human AR spermatozoa induced with calcium ionophore A23187 were used for immunization, and the hybridoma secreting monoclonal antibody (MoAb) to non-treated (NT) and AR human sperm was screened with enzyme-linked immunosorbent assay (ELISA). Twenty three MoAbs have been obtained, and immunochemical, immunocytochemical, biochemical and functional characteristics of these MoAbs and their antigens have been evaluated. The results are briefly shown as follows: 1. By using ELISA 12 MoAbs (A group) and 6 MoAbs (B group) reacted mainly with the AR and NT sperm antigens, respectively, and 5 MoAbs (C group) did with both sperm. 2. Of 23 MoAbs, 21 MoAbs were classified as IgM, others as IgG_1 and IgG_(2e) by Ouchterlony test. 3. The cross reaction of 23 MoAbs with human white cell lines were detected and the discrepant reactions among the MoAbs were shown. 4. The antigens recognized by most of MoAbs are located in equatorial segment and midpiece of human sperm, and no one in acrosome region of human sperm. The binding pattern of some MoAbs on NT and AR sperm were found to be different. These results suggest that the immunogenicity may be different between the AR sperm and the NT sperm. 5. The strong sperm agglutination were caused by 9 MoAbs. 6. The human sperm proteins ranged in the size of 16-146 kDa were demonstrated by immunoblotting with 9 MoAbs. The further characterization of these MoAbs and their antigens is in progress. Ten MoAbs were submitted to the human sperm antigen classification program organized by World Health Organization. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6227]  |
| 专题 | 昆明动物研究所_其他 |
| 推荐引用方式 GB/T 7714 | 王云美. 抗顶体反应人精子单克隆抗体的制备和鉴定[D]. 北京. 中国科学院研究生院. 1991. |
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