Hemati Atefeh, Azarnia Mahnaz, Hossein Modarressi Mohammad, Rahimi Amir
Department of Cell and Molecular Biology, School of Biological Sciences, Kharazmi University, Tehran, Iran.
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Hum Antibodies. 2018;26(4):209-218. doi: 10.3233/HAB-180340.
Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field.
Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field.
To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot.
By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI.
So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.
据报道，针对睾丸抗原产生并利用特异性单克隆抗体可鉴定在生殖领域中重要的抗原。
当前研究旨在引入一种产生特异性抗睾丸单克隆抗体的杂交瘤，以鉴定在生殖领域可能重要的睾丸抗原。
为制备针对睾丸抗原的杂交瘤，用睾丸细胞裂解物免疫小鼠。细胞融合后，通过间接ELISA筛选所得杂交瘤，然后通过有限稀释法进行克隆，最后用免疫组织化学、免疫细胞化学和蛋白质印迹等一些分子实验室技术对产生的单克隆抗体进行表征。
通过杂交瘤技术进行细胞融合，最终产生了10种（8A11、8D6、8D7、9F6、9G11、10C3、10B3、10B2、10C2和10H7）针对睾丸抗原的特异性抗体。在产生 的抗体中，发现10C3与睾丸发生交叉反应，但在其他组织中未检测到。单克隆抗体10C3识别精子和睾丸抗原，特别是睾丸间质组织、精原细胞、初级和次级精母细胞抗原，因此它们很可能是所产生单克隆抗体的靶标。此外，我们的单克隆抗体能够完全检测小鼠精子抗原以及人类精子头部和尾部的特异性抗原。在蛋白质印迹分析中，单克隆抗体10C3能够识别精子和睾丸提取物的特异性蛋白条带。此外，在本研究中，根据NCBI的UniGene部分提供的小鼠EST图谱选择了作为所产生单克隆抗体靶标的睾丸特异性基因。
因此，这种稳定的抗睾丸单克隆抗体在实验室研究以及受精诊断程序中具有潜力。因此，它可被用作几种疾病的靶标特异性诊断和研究的合适工具。
