利用精子特异性抗体SPAG8分离混合细胞体系中的精子

发布时间：2018-03-13 23:38

本文选题：SPAG　切入点：免疫磁珠　出处：《第三军医大学学报》2014年12期 　论文类型：期刊论文

【摘要】：目的利用精子特异性抗体SPAG8(Sperm associated antigen 8)结合免疫磁珠技术分离混合细胞系中的精子,建立混合斑中精子细胞分离的新方法。方法制备含有精子103、104、105/mL与阴道上皮细胞104/mL的3种混合悬液各10例,各样本制作混悬液及模拟混合斑各2份,分别以生物素标记的SPAG8单克隆抗体孵育特异结合混合悬液及混合斑中的精子,然后用亲和素包被的免疫磁珠通过生物素-亲和素复合体捕获分离精子细胞,提取精子DNA进行STR复合扩增和DNA分型检测,同时以差异裂解法检测混悬液及混合斑中的精子进行方法比较。结果免疫磁珠-亲和素-生物素-抗体复合物可分离混合细胞体系中的精子,当混合悬液内的精子数量达到103/mL时,90%的样本可得到13个STR基因座的单一男性分型,精子数量达到104/mL时,90%的样本均可得到16个STR基因座的单一男性分型;含有104/mL以上数量精子的混悬液制作的混合斑,单一男性13个STR基因座的正确分型率达80%以上,混悬液及混合斑的免疫磁珠法有效STR检出率高于传统的差异裂解法。结论本方法简便快捷,可作为混合斑法医学检验的有效补充手段。
[Abstract]:Objective to isolate spermatozoa from mixed cell lines by sperm specific antibody SPAG8(Sperm associated antigen 8 and immunomagnetic beads. A new method for the separation of sperm cells from mixed plaque was established. Methods three kinds of mixed suspensions containing 103X 104C / mL spermatozoa and 104 / mL vaginal epithelial cells were prepared. The spermatozoa were incubated with biotinylated SPAG8 monoclonal antibodies (McAbs), respectively, and spermatozoa in mixed suspension and mixed spot were incubated with avidin coated immunomagnetic beads, which were captured and separated by biotin-affinity complex. Sperm DNA was extracted for STR amplification and DNA typing. At the same time, the method of differential cleavage was used to detect the spermatozoa in suspension and mixed spot. Results the immunomagnetic bead avidin biotin antibody complex could separate the sperm from the mixed cell system. When the number of spermatozoa in mixed suspension was 103 / mL, the single male typing of 13 STR loci could be obtained in 90% of the samples, and the single male typing of 16 STR loci could be obtained in 90% samples with a sperm count of 104% / mL. The correct typing rate of 13 STR loci in a single male was more than 80% for the mixed spots made by suspension containing more than 104% mL of sperm. The detectable rate of STR by immunomagnetic bead method is higher than that by traditional differential cleavage method. Conclusion this method is simple and rapid and can be used as an effective supplementary method for medical examination of mixed plaque.
【作者单位】：山东省高校证据鉴识重点实验室(山东政法学院);重庆市公安局物证鉴定中心;重庆市公安局江北分局法医科;
【基金】：山东省高校证据鉴识重点实验室(山东政法学院)开放课题(2014-01) 重庆市公安局科技攻关项目(2012-05)~~
【分类号】：D919.4

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