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人血清中非Gal异种抗体检测方法的探讨

叶学军 卢希彬 潘登科 蔡志明 牟丽莎 赵成江

叶学军, 卢希彬, 潘登科, 等. 人血清中非Gal异种抗体检测方法的探讨[J]. 器官移植, 2017, 8(2): 132-137. doi: 10.3969/j.issn.1674-7445.2017.02.008
引用本文: 叶学军, 卢希彬, 潘登科, 等. 人血清中非Gal异种抗体检测方法的探讨[J]. 器官移植, 2017, 8(2): 132-137. doi: 10.3969/j.issn.1674-7445.2017.02.008
Ye Xuejun, Lu Xibin, Pan Dengke, et al. Detection methods of non-Gal xenoantibody in human serum[J]. ORGAN TRANSPLANTATION, 2017, 8(2): 132-137. doi: 10.3969/j.issn.1674-7445.2017.02.008
Citation: Ye Xuejun, Lu Xibin, Pan Dengke, et al. Detection methods of non-Gal xenoantibody in human serum[J]. ORGAN TRANSPLANTATION, 2017, 8(2): 132-137. doi: 10.3969/j.issn.1674-7445.2017.02.008

人血清中非Gal异种抗体检测方法的探讨

doi: 10.3969/j.issn.1674-7445.2017.02.008
基金项目: 

深圳市高水平医学学科建设专项基金 2016031638

深圳市三名工程;深圳市科创委学科布局项目 JCYJ20160229204849975

深圳市科创委企业工程中心项目 GCZX2015043017281705

深圳市卫计委临床技术研究与转化类 201501018

详细信息
    通讯作者:

    牟丽莎, Email:molly__molly@163.com

    赵成江, Email:charleszhao423@gmail.com

  • 中图分类号: R-332, R617

Detection methods of non-Gal xenoantibody in human serum

More Information
  • 摘要:   目的  探讨人血清中抗非半乳糖(Gal)异种抗原抗体的最佳检测条件。  方法  选用α-1, 3半乳糖基转移酶基因敲除(GTKO)五指山小型猪的外周血单核细胞(PBMC) 作为靶细胞,与健康人血清混合,在不同血清浓度(4.8%、16.7%、100%)和孵育时间(0.5、1.0、2.0、3.0、6.0 h)条件下,利用流式细胞仪检测GTKO猪的PBMC上结合IgM和IgG的水平。  结果  在16.7%血清浓度下,孵育时间从0.5 h延长至3.0 h,能显著提高非Gal IgM结合PBMC的水平(P < 0.01),而IgG水平的提高则差异无统计学意义(P > 0.05)。提高血清浓度也可提高非Gal IgM的结合水平,采用100%的血清浓度孵育3 h,IgM结合PBMC水平最高且有统计学意义(P < 0.01)。采用100%的血清孵育6 h,IgG结合水平升高有统计学意义(P < 0.05)。延长孵育时间和提高血清浓度不会影响PBMC的活力。  结论  检测人血清中抗非Gal异种抗原抗体的最佳条件为每1×105个猪PBMC,采用100%人血清浓度孵育3 h检测IgM水平,或加100%人血清浓度孵育6 h检测IgG水平。这一条件的优化有助于筛选非Gal表达抗原低表达的供体猪。

     

  • 图  1  猪αGal表型的鉴定

    A图为BSI-B4对猪全血的凝集反应,左图为野生型,右图为GTKO型;B图为流式细胞仪检测BSI-B4与野生型PBMC的结合;C图为流式细胞仪检测BSI-B4与GTKO型PBMC的结合

    Figure  1.  Identification of porcine αGal phenotype

    图  2  不同孵育时间对非Gal异种抗体IgM或IgG结合的影响

    A图血清浓度4.8%,B图血清浓度16.7%,C图血清浓度100%;aP < 0.01

    Figure  2.  Effect of different incubation time on the binding of non Gal IgM or IgG xenoantibody

    图  3  不同血清浓度对非Gal异种抗体IgM结合水平的影响

    aP <0.05, bP < 0.01

    Figure  3.  Effect of different serum concentration on the binding of non Gal IgM

    图  4  不同浓度血清对对非Gal异种抗体IgG结合水平的影响

    aP <0.05

    Figure  4.  Effect of different serum concentration on the binding of non Gal IgG

    表  1  近10年间利用猪PBMC检测人血清中IgM和IgG抗体结合实验所使用的血清浓度和孵育时间

    Table  1.   Detection of serum concentrations and incubation time of IgM and IgG antibodies combined experiment in human serum by porcine PBMC in recent ten years

    细胞基因型 血清浓度 孵育时间(h) 文献 发表年份
    GTKO、GTKO/iGb3sKO 25% 1.0 Butler JR, et al[5] 2016
    GTKO/NeuGCKO、GTKO/NeuGCKO/hCD55、GTKO/NeuGCKO/B4GalKO 25% 1.0 Butler JR, et al[6] 2016
    GTKO、GTKO/NeuGcKO、GTKO/NeuGCKO/β4GalKO 25% 0.5 Estrada JL, et al[7] 2015
    GTKO、GTKO/NeuGCKO 25% 未列出 Burlak C, et al[8] 2014
    GTKO、GTKO/NeuGCKO 25% 2.0 Lutz AJ, et al[9] 2013
    WT、GTKO/CD46、GTKO/NeuGCKO/hCD46 20% 0.5 Lee W, et al[10] 2016
    WT、GTKO、GTKO/hCD46、hCD46 20% 0.5 Hara H, et al[11] 2008
    WT、GTKO 20% 0.5 Rood PP, et al[12] 2007
    WT、GTKO 20% 0.5 Hara H, et al[13] 2006
    WT、GTKO 20% 0.5 Rood PP, et al[14] 2006
    WT、GTKO 20% 0.5 Ezzelarab M, et al[15] 2006
    GTKO 10% 0.5 Liang F, et al[16] 2013
    GTKO 9% 0.5 Wong BS, et al[17] 2006
    下载: 导出CSV
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    [8] Burlak C, Paris LL, Lutz AJ, et al. Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs[J]. Am J Transplant, 2014, 14(8):1895-1900. DOI: 10.1111/ajt.12744.
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    [12] Rood PP, Tai HC, Hara H, et al. Late onset of development of natural anti-nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants[J]. Transpl Int, 2007, 20(12):1050-1058. doi: 10.1111/tri.2007.20.issue-12
    [13] Hara H, Ezzelarab M, Rood PP, et al. Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans[J]. Xenotransplantation, 2006, 13(4):357-365. doi: 10.1111/xen.2006.13.issue-4
    [14] Rood PP, Hara H, Busch JL, et al. Incidence and cytotoxicity of antibodies in cynomolgus monkeys directed to nonGal antigens, and their relevance for experimental models[J]. Transpl Int, 2006, 19(2):158-165. doi: 10.1111/tri.2006.19.issue-2
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    [16] Liang F, Wamala I, Scalea J, et al. Increased levels of anti-non-Gal IgG following pig-to-baboon bone marrow transplantation correlate with failure of engraftment[J]. Xenotransplantation, 2013, 20(6):458-468. DOI: 10.1111/xen.12065.
    [17] Wong BS, Yamada K, Okumi M, et al. Allosensitization does not increase the risk of xenoreactivity to alpha1, 3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists[J]. Transplantation, 2006, 82(3):314-319. doi: 10.1097/01.tp.0000228907.12073.0b
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出版历程
  • 收稿日期:  2016-12-28
  • 网络出版日期:  2021-01-19
  • 刊出日期:  2017-03-15

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