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
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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10~3/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≥10~4/mL时,分型成功率达10~0%;对精子数分别为10~3/mL、10~4/mL、10~5/mL的混合斑STR分型成功率分别为40%、90%和10~0%。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。
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 m Ab 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 10~3/m L and 10~0% when the sperm cells number was equal to or more than 10~4/m L.When the number of sperm cells was 10~3/m L,10~4/m L and 10~5/m L in mixed stain samples,the success rate of STR typing were 40%,90% and 10~0%,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.
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 m Ab 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 10~3/m L and 10~0% when the sperm cells number was equal to or more than 10~4/m L.When the number of sperm cells was 10~3/m L,10~4/m L and 10~5/m L in mixed stain samples,the success rate of STR typing were 40%,90% and 10~0%,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.
引文
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[25]Anslinger K,Bayer B,Danilov SM,et al.Application of sperm-specific antibodies for the separation of sperm from cell mixtures.Foren Sci Int:Genet Suppl Ser,2008,1(1):394-395.
[26]Zhang EL,Dong JL,Liu L,et al.Magnetic bead-based directional capture of sperm using a polyclonal antibody against ADAM2.Life Sci Instr,2014,12(6):34-36,48(in Chinese).张尔力,董军磊,刘琳,等.抗ADAM2抗体免疫磁珠捕获精子的新方法研究.生命科学仪器,2014,12(6):34-36,48.
[27]Li XB,Wang QS,Ning SH,et al.Separation of sperms from cell mixtures by sperm-specific antibody SPAG8.JThird Mil Med Univ,2014,36(12):1322-1326(in Chinese).李学博,王清山,宁淑华,等.利用精子特异性抗体SPAG8分离混合细胞体系中的精子.第三军医大学学报,2014,36(12):1322-1326.
[28]Willott GM,Allard JE,Spermatozoa-their persistence after sexual intercourse.Foren Sci Int,1982,19(2):135-154.
[2]Voskoboinik L,Darvasi A.Forensic identification of an individual in complex DNA mixtures.Forensic Sci Int Genet,2011,5(5):428-435.
[3]Lei L,Zang LL,Xu J,et al.Research advances of mixed stains in forensic science.Chin J Forensic Med,2015,30(3):273-276(in Chinese).雷亮,臧丽丽,徐洁,等.法医学混合斑研究进展.中国法医学杂志,2015,30(3):273-276.
[4]Hennekens CM,Cooper ES,Cotton RW,et al.The effects of differential extraction conditions on the premature lysis of spermatozoa.J Forensic Sci,2013,58(3):744-752.
[5]Garvin AM.Filtration based DNA preparation for sexual assault cases.J Forensic Sci,2003,48(5):1084-1087.
[6]Budimlija ZM,Lechpammer M,Popiolek D,et al.Forensic applications of laser capture microdissection:use in DNA-based parentage testing and platform validation.Croat Med J,2005,46(4):549-555.
[7]Schoell WM,Klintschar M,Mirhashemi R,et al.Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault.Obstet Gynecol,1999,94(4):623-627.
[8]Klein SB,Buoncristiani MR.Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.Forensic Sci Int Genet,2017,29:109-117.
[9]Vandewoestyne M,Deforce D.Laser capture microdissection in forensic research:a review.Int JLegal Med,2010,124(6):513-521.
[10]Li CX,Han JP,Ren WY,et al.DNA profiling of spermatozoa by laser capture microdissection and low volume-PCR.PLoS ONE,2011,6(8):e22316.
[11]Horsman KM,Barker SLR,Ferrance JP,et al.Separation of sperm and epithelial cells in a microfabricated device:potential application to forensic analysis of sexual assault evidence.Anal Chem,2005,77(3):742-749.
[12]Liu WR,Chen WX,Liu R,et al.Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis.Biomicrofluidics,2015,9(4):044127.
[13]Li XB,Wang QS,Feng Y,et al.Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.Int J Legal Med,2014,128(6):905-911.
[14]Zhao XC,Wang L,Sun J,et al.Isolating sperm from cell mixtures using magnetic beads coupled with an anti-PH-20 antibody for forensic DNA analysis.PLoSONE,2016,11(7):e0159401.
[15]Hamil KG,Liu Q,Sivashanmugam P,et al.LCN6,a novel human epididymal lipocalin.Reprod Biol Endocrinol,2003,1(1):112-125.
[16]Chen J,Feng W,Zhao Y,et al.Expression,purification,and refolding of human lipocalin 6 and production of a monoclonal antibody against this protein.Monoclon Antib Immunodiagn Immunother,2017,36(4):185-191.
[17]Liddell JE,Cryer A.A Practical Guide to Monoclonal Antibodies.Chichester:John Wiley&Sons,Inc.,1991:110-115.
[18]Guan XY,Xu ZC,Wang YQ,et al.Generation and characterization of monoclonal antibodies against chicken interleukin 4.Chin J Biotech,2017,33(1):44-54(in Chinese).关晓宇,徐志超,王永强,等.抗鸡白细胞介素4单克隆抗体的制备及鉴定.生物工程学报,2017,33(1):44-54.
[19]Luo JM,Zheng YL,Zhou YC,et al.Identification of sperm-binding proteins in the ventral prostate of the golden hamster.Nat J Androl,2007,13(10):883-889(in Chinese).骆建民,郑玉鸾,周元聪,等.金黄地鼠腹侧前列腺来源蛋白结合精子表面的实验研究.中华男科学杂志,2007,13(10):883-889.
[20]Butler JM.DNA mixtures//Advanced Topics in Forensic DNA Typing:Interpretation.San Diego:Elsevier Inc.,2014:32-41.
[21]Li XJ,Han JL,Li L,et al.Research advances of separation methods of sperm cells from mixtures.JKunming Med Univ,2016,37(2):140-143(in Chinese).李秀江,韩建利,李丽,等.混合斑中精子细胞的分离方法研究进展.昆明医科大学学报,2016,37(2):140-143.
[22]?afa?ík I,?afa?íkva M.Use of magnetic techniques for the isolation of cells.J Chromatogr B:Biomed Sci Appl,1999,722(1/2):33-53.
[23]Lei JH,Guan F,Xu H,et al.Application of an immunomagnetic bead ELISA based on IgY for detection of circulating antigen in urine of mice infected with Schistosoma japonicum.Vet Parasitol,2012,187(1/2):196-202.
[24]Arthur E.Spermatozoa capture during the differential extraction process for STR typing of sexual assault evidence.US,2000-IJ-CX-K009.2002-02.
[25]Anslinger K,Bayer B,Danilov SM,et al.Application of sperm-specific antibodies for the separation of sperm from cell mixtures.Foren Sci Int:Genet Suppl Ser,2008,1(1):394-395.
[26]Zhang EL,Dong JL,Liu L,et al.Magnetic bead-based directional capture of sperm using a polyclonal antibody against ADAM2.Life Sci Instr,2014,12(6):34-36,48(in Chinese).张尔力,董军磊,刘琳,等.抗ADAM2抗体免疫磁珠捕获精子的新方法研究.生命科学仪器,2014,12(6):34-36,48.
[27]Li XB,Wang QS,Ning SH,et al.Separation of sperms from cell mixtures by sperm-specific antibody SPAG8.JThird Mil Med Univ,2014,36(12):1322-1326(in Chinese).李学博,王清山,宁淑华,等.利用精子特异性抗体SPAG8分离混合细胞体系中的精子.第三军医大学学报,2014,36(12):1322-1326.
[28]Willott GM,Allard JE,Spermatozoa-their persistence after sexual intercourse.Foren Sci Int,1982,19(2):135-154.