hLCN6单克隆抗体偶联免疫磁珠用于混合细胞中精子的分离与法医学鉴定

Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads

摘要:

hLCN6 (Human lipocalin 6) 是附睾特异性分泌蛋白，它能与精子结合，对精子的成熟发挥着重要作用。为了探究应用抗hLCN6单克隆抗体偶联免疫磁珠技术分离混合细胞中精子的可行性，建立混合斑中精子细胞分离的新方法，制备了不同比例的精子-上皮细胞混合悬液及混合斑样本，以生物素标记的hLCN6单克隆抗体孵育样品，再用亲和素包被的免疫磁珠捕获分离精子细胞，提取精子DNA进行PCR-STR (Short tandem repeat) 分型，同时以差异裂解法提取精子，比较两者的差异。经ELISA检测，hLCN6单克隆抗体与抗原的亲和力解离常数 (Kd) 为3.47×10–9 mol/L。免疫印迹和免疫荧光结果显示，hLCN6在精子中可检测到，主要定位于精子头部的顶体后区域，但不能在上皮细胞中检测到。hLCN6抗体偶联的免疫磁珠复合物能够实现精子细胞的捕获和分离，显微镜观察显示免疫磁珠能够与精子头部特异性结合。对精子个数为103/mL的混合悬液，STR分型成功率 (正确分型13个以上，RFU>200) 为90%。当精子数量≥104/mL时，分型成功率达100%；对精子数分别为103/mL、104/mL、105/mL的混合斑STR分型成功率分别为40%、90%和100%。综上，hLCN6抗体偶联免疫磁珠法可以有效分离混合细胞中的精子，分型成功率高于传统的差异裂解法。该方法简单高效，可作为性侵案件中法医混合斑检验的有效补充手段。

Abstract:

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10–9 mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 103/mL and 100% when the sperm cells number was equal to or more than 104/mL. When the number of sperm cells was 103/mL, 104/mL and 105/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.