[1]宋敏,范娅涵,王世春,等.间接ELISA测定人血IgM型抗-A抗体方法的建立及应用[J].第三军医大学学报,2018,40(07):556-560.
 SONG Min,FAN Yahan,WANG Shichun,et al.Establishment and application of an indirect ELISA for determining IgM anti-A antibodies in human plasma[J].J Third Mil Med Univ,2018,40(07):556-560.
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间接ELISA测定人血IgM型抗-A抗体方法的建立及应用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第07期
页码:
556-560
栏目:
基础医学
出版日期:
2018-04-15

文章信息/Info

Title:
Establishment and application of an indirect ELISA for determining IgM anti-A antibodies in human plasma
作者:
宋敏范娅涵王世春张强徐婷蒋天伦赵树铭
陆军军医大学(第三军医大学)第一附属医院输血科
Author(s):
SONG Min FAN Yahan WANG Shichun ZHANG Qiang XU Ting JIANG Tianlun ZHAO Shuming

Department of Blood Transfusion, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
ABO血型血影血型抗体定量ELISA
Keywords:
ABO blood group erythrocyte ghosts blood group antibody quantitation ELISA
分类号:
R392-33; R392.11; R457.11
文献标志码:
A
摘要:

目的     建立间接酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测方法,快速定量检测人血IgM抗-A抗体。方法     通过制备A型红细胞膜蛋白作为捕获抗原,采用棋盘滴定实验筛选出最佳抗原抗体工作浓度,建立人血IgM型抗-A抗体定量检测方法。结果     所构建的间接ELISA定量检测方法检测下限为0.02 μg/mL;批内变异系数及批间变异系数均小于10%;通过ROC曲线分析,当临界D(450)为0.259时,ROC曲线下面积最大,为0.947,检测灵敏度为87.7%,特异性为100%。采用本研究自建的ELISA检测57例B型血浆标本的IgM型抗-A抗体浓度为(1.71±1.95)μg/mL,定量结果与经典半定量效价评分呈正相关(r=0.71,P<0.01)。结论     成功建立了人血IgM型抗A抗体定量检测方法。

Abstract:

Objective    To develop an indirect enzyme linked immunosorbent assay (ELISA) for rapid and quantitative detection of IgM anti-A antibodies in human plasma. Methods     An in-house indirect ELISA was developed using the ghost protein of group A erythrocyte as coating antigen. The optimal combination of antigens and antibodies was selected by checkerboard titration. The methodological performance was evaluated, and the plasma concentrations of IgM anti-A antibodies of clinical samples were determined. Results     The detection limit of the in-house indirect ELISA was 0.02 μg/mL. The coefficient of variation for inner-batch and inter-batch were both less than 10%. According to the receiver operating characteristic (ROC) curve analysis, the largest area under the curve accounted for 0.947 when the cut-off value at D(450) was 0.259, and the detection sensitivity and specificity were 87.7% and 100% respectively. The plasma level of IgM anti-A antibodies in 57 samples of type B plasma was 1.71±1.95 μg/mL. The quantitative results were positively correlated to the titer scores from the classical semi-quantitative detection of IgM anti-A antibodies (r=0.71, P<0.01). Conclusion    An indirect ELISA is developed successfully for quantitative detection of IgM antiA antibodies.

参考文献/References:

[1]COOLING L. ABO and platelet transfusion therapy[J]. Immunohematology, 2007, 23 (1): 20-33.
[2]FUNG M K, DOWNES K A, SHULMAN I A. Transfusion of platelets containing ABOincompatible plasma: a survey of 3 156 North American laboratories[J]. Arch Pathol Lab Med, 2007, 131 (6): 909-916. DOI: 10.1043/15432165(2007)131[909:TOPCAP]2.0.CO;2.
[3]HARRIS S B, JOSEPHSON C D, KOST C B, et al. Nonfatal intravascular hemolysis in a pediatric patient after transfusion of a platelet unit with high-titer anti-A[J]. Transfusion, 2007, 47 (8): 1412-1417. DOI: 10.1111/j.1537-2995.2007.01283.x.
[4]KOBAYASHI T, SAITO K. A series of surveys on assay for anti-A/B antibody by Japanese ABOincompatible Transplantation Committee[J]. Xenotransplantation, 2006, 13(2): 136-140. DOI: 10.1111/j.1399-3089. 2006.00296.x.
[5]PESCIOTTA E N, LAM H S, KOSSENKOV A, et al. In-depth, label-free analysis of the erythrocyte cytoplasmic proteome in Diamond Blackfan anemia identifies a unique inflammatory signature[J]. PLoS ONE, 2015, 10 (10): e140036. DOI: 10.1371/journal.pone.0140036.
[6]CROWTHER J R. The ELISA guidebook [M]. 2nd ed. New York: Humana Press, 2009. 566.
[7]LEE M F, LIN T M, LIU S W, et al. A rapid method of detecting autoantibody against FcepsilonRIalpha for chronic spontaneous urticaria[J]. PLoS ONE, 2014, 9(10): e109565. DOI: 10.1371/journal.pone.0109565.
[8]DENG Q, QIU M, WANG Y, et al. A sensitive and validated immunomagnetic-bead based enzymelinked immunosorbent assay for analyzing total T-2 (free and modified) toxins in shrimp tissues[J]. Ecotoxicol Environ Saf, 2017, 142: 441-447. DOI: 10.1016/j.ecoenv. 2017.04.037.
[9]兰炯采,吴涛,张国鹏,等. 用ELISA双抗夹心法检测sHLA-Ⅰ在不同贮存成分血中的浓度[J]. 中国输血杂志, 2003, 16(6): 402-404. DOI: 10.3969/j.issn.1004-549X.2003.06.007.
LAN J C, WU T, ZHANG G P, et al. Detection of soluble HLA-Ⅰconcentration in blood components at different storage by ELISA double antigen sandwich assay[J]. Chin J Blood Transfus, 2003, 16 (6): 402-404.
[10]中华人民共和国卫生部医政司. 全国临床检验操作规程[M].3版. 南京:东南大学出版社,2006: 791-798.
Department of Medical Administration of Ministry of Health of People’s Republic of China. National guide to clinical laboratory prodedures [M]. 3 ed. Nanjing: Southeast University Press, 2006: 791-798.
 [11]NORRIS J M, DIKEMAN J, GREENE D, et al. Managing change[J]. Transfusion, 2009, 49 (11 Pt 2): 2524-2535. DOI: 10.1111/j.1537-2995. 2009.02470.x.
[12]MORIOKA D, TOGO S, KUMAMOTO T, et al. Six consecutive cases of successful adult ABOincompatible living donor liver transplantation: a proposal for grading the severity of antibody-mediated rejection[J]. Transplantation, 2008, 85(2): 171-178. DOI: 10.1097/TP.0b013e31815 e9672.
[13]BELLAMY C O, HERRIOT M M, HARRISON D J, et al. C4d immunopositivity is uncommon in ABOcompatible liver allografts, but correlates partially with lymphocytotoxic antibody status[J]. Histopathology, 2007, 50 (6): 739-749. DOI: 10.1111/j.1365-2559.2007.02677.x.
[14]YURUGI K, KIMURA S, ASHIHARA E, et al. Rapid and accurate measurement of antiA/B IgG antibody in ABOunmatched living donor liver transplantation by surface plasmon resonance[J]. Transfus Med, 2007, 17 (2): 97-106. DOI: 10.1111/j.1365-3148.2007.00737.x.
[15]SATOH A, KAWAGISHI N, MINEGISHI M, et al. Development of a novel ELISA for detection of antiA and anti-B antibodies in recipients of ABO-incompatible living donor liver grafts[J]. Tohoku J Exp Med, 2007, 211(4): 359-367. DOI: 10.1620/tjem.211.359.
[16]张敏,芦慧霞,辛琪. IgG抗-A/BELISA检测方法的初探[J]. 中国输血杂志, 2011,24(5): 414-415. DOI: 10.13303/j.cjbt.issn.1004549x.2011.05.018.
ZHANG M, LU H X, XIN Q. Primary investigation of ELISA for determination of IgG antiA/B[J]. Chin J Blood Transfus, 2011, 24(5): 414-415. DOI: 10.13303/j.cjbt.issn.1004549x.2011.05.018.
[17]卢新奇,张志峰. 定量测定ABO血型IgG抗体新方法的研究[J]. 医学检验与临床, 2012, 23(1): 72-73. DOI: 10.3969/j.issn.1673-5013.2012.01.031.
LU X Q, ZHANG Z F. A new method for quantitative determination of the ABO blood group IgG antibody[J]. Med Lab Sci Clin, 2012, 23 (1): 72-73. DOI: 10.3969/j.issn.1673-5013.2012.01.031.
[18]SPALTER S H, KAVERI S V, BONNIN E, et al. Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens[J]. Blood, 1999, 93 (12): 4418-4424.

更新日期/Last Update: 2018-04-09