Human lipocalin 6 (hLCN6) is a newly discovered epididymal-specific secreted protein, capable of binding to the head and tail of spermatozoa and involved in sperm maturation. Anti-hLCN6 monoclonal antibody coupled immunomagnetic beads (IMBs) can be effectively used for the separation and forensic identification of sperm cells from mixed stains. But the source of monoclonal antibody is limited. In this study, an immunized mouse phage display antibody library was constructed and the single-chain variable fragments (scFvs) against hLCN6 were screened. The selection was performed using four rounds of biopanning and positive clones were validated by phage ELISA. Two anti-hLCN6 scFv clones with highest affinity were selected and sequencing result showed that the two sequences were identical. After prokaryotic expression and purification, the purified scFv could specifically recognize the hLCN6 in the lysate of human sperm cells and epididymis by western blot analysis, without any cross-reactivity with cellular antigens in female epithelial cells. The dissociation constant (Kd) of anti-hLCN6 scFv was 6.69 × 10-7 mol/L measured by indirect ELISA. Therefore, our work not only provides a useful tool for further exploration of the biological functions of hLCN6, but also opens up new research avenues for the separation of sperm cells from mixed stains based on immuno-binding reaction.

中文翻译：

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通过噬菌体展示技术选择，制备和鉴定针对人脂钙素6的scFv。

人lipocalin 6（hLCN6）是新发现的附睾特异性分泌蛋白，能够与精子的头和尾结合并参与精子的成熟。抗hLCN6单克隆抗体偶联免疫磁珠（IMB）可有效地用于从混合污渍中分离和法医鉴定精子细胞。但是单克隆抗体的来源是有限的。在这项研究中，构建了免疫的小鼠噬菌体展示抗体文库，并筛选了针对hLCN6的单链可变片段（scFvs）。使用四轮生物淘选进行选择，并通过噬菌体ELISA验证阳性克隆。选择了两个亲和力最高的抗hLCN6 sc​​Fv克隆，测序结果表明两个序列相同。经过原核表达纯化 纯化的scFv可以通过Western印迹分析特异性识别人精子细胞和附睾裂解物中的hLCN6，而与女性上皮细胞中的细胞抗原没有任何交叉反应。通过间接ELISA测定的抗hLCN6 sc​​Fv的解离常数（Kd）为6.69×10-7 mol / L。因此，我们的工作不仅为进一步探索hLCN6的生物学功能提供了有用的工具，而且为基于免疫结合反应从混合污渍中分离精子细胞开辟了新的研究途径。