HPA-1a、HPA-3a、HPA-5b抗体MAIPA定量检测方法的建立

doi:10.3969/j.issn.1673-8640.2018.01.009

摘要/Abstract

摘要：

目的分别建立针对人类血小板抗原（HPA）-1a、HPA-3a、HPA-5b 3种抗体的定量血小板（PLT）抗原单克隆抗体特异性免疫固定检测（MAIPA）方法。方法采用MAIPA方法检测不同浓度的标准或参比HPA-1a、HPA-3a、HPA-5b抗血清,优化实验条件,在检测范围内绘制线性曲线,并分别分析其敏感性、特异性、准确性和精密度。结果针对HPA-1a、HPA-3a、HPA-5b 3种抗体的MAIPA定量检测方法得到了优化,检测范围分别为0~4 IU、0~20 AU、0~20 AU,准确性（回收率）分别为96.20%~103.10%、97.90%~103.10%、100.20%~106.10%。批内精密度[变异系数（CV）]分别为1.3%~3.6%、2.7%~5.2%和4.7%~5.3%,批间精密度（CV）分别为4.0%~5.6%、4.4%~5.9%和3.2%~6.3%,检测人源抗体抗A、抗B、抗I、抗P均无明显阳性,具有良好的特异性。结论建立的针对HPA-1a、HPA-3a、HPA-5b抗体的MAIPA定量检测体系均具有较高的敏感度、特异度、准确度和精密度,对于临床相关疾病的诊断和PLT安全输注具有重要的应用价值。

关键词: 人类血小板抗原, 单克隆抗体特异性免疫固定检测, 定量, 标准体系

Abstract:

Objective To establish quantitative monoclonal antibody-specific immobilization of platelet（PLT） antigen （MAIPA） assay for human PLT antigen（HPA）-1a,HPA-3a and HPA-5b antibodies.Methods MAIPA assay was applied on serial dilutions of standard or reference reagents containing antibodies against HPA-1a,HPA-3a and HPA-5b. Optimal experimental parameters were determined,and standard curves were constructed with linear regression. The sensitivity,specificity and reproducibility of the assay were evaluated. Results The optimal reagents and conditions for MAIPA assay were decided. The determination ranges were 0-4 IU,0-20 AU and 0-20 AU,and accuracies（recovery rates）were 96.20%-103.10%,97.90%-103.10% and 100.20%-106.10%. The within-run precisions [coefficients of variation （CV）] for HPA-1a,HPA-3a and HPA-5b antibodies were 1.3%-3.6%,2.7%-5.2% and 4.7%-5.3%. The between-run precisions （CV） were 4.0%-5.6%,4.4%-5.9% and 3.2%-6.3%. There was no positivity for anti-A,anti-B,anti-I and anti-P humanized antibodies with good specificities.Conclusions The established quantitative MAIPA assay for HPA-1a,HPA-3a and HPA-5b antibodies has good sensitivity,specificity,accuracy and precision. It plays a role in the clinical diagnosis of alloimmunization disease caused by HPA antibodies and safe transfusion of PLT.

Key words: Human platelet antigen, Monoclonal antibody-specific immobilization of platelet antigen, Quantitation, Standard system

中图分类号:

R446.1

李睿书, 凌冰, 陆萍. HPA-1a、HPA-3a、HPA-5b抗体MAIPA定量检测方法的建立[J]. 检验医学, 2018, 33(1): 50-54.

LI Ruishu, LING Bing, LU Ping. Establishment of quantitative MAIPA assay for HPA-1a,HPA-3a and HPA-5b antibodies[J]. Laboratory Medicine, 2018, 33(1): 50-54.

图/表 5

参考文献 17

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待测抗体	HPA-1a	HPA-3a	HPA-5b
小鼠单克隆抗体	鼠抗人 CD61（美国BD 公司,批号555752）	抗人整合素α2b/CD41（美国R&D公司,批号MAB7616）	抗人整合素 α2/CD49b（美国R&D公司,批号MAB1233）
小鼠单克隆抗体工作浓度（μg/mL）	20	2	1
羊抗鼠IgG工作浓度	1∶10 000	1∶3 000	1∶1 000

待测抗体	HPA-1a	HPA-3a	HPA-5b
小鼠单克隆抗体	鼠抗人 CD61（美国BD 公司,批号555752）	抗人整合素α2b/CD41（美国R&D公司,批号MAB7616）	抗人整合素 α2/CD49b（美国R&D公司,批号MAB1233）
小鼠单克隆抗体工作浓度（μg/mL）	20	2	1
羊抗鼠IgG工作浓度	1∶10 000	1∶3 000	1∶1 000

项目	HPA-1a（IU）	HPA-3a（AU）	HPA-5b（AU）
x	0.231	0.314	0.377
s	0.027	0.309	0.034
x +3s	0.312	0.406	0.477
敏感性^*	0.3	1.0	1.5

项目	HPA-1a（IU）	HPA-3a（AU）	HPA-5b（AU）
x	0.231	0.314	0.377
s	0.027	0.309	0.034
x +3s	0.312	0.406	0.477
敏感性^*	0.3	1.0	1.5

项目	批内			批间
项目	x	回收率（%）	CV（%）	x	回收率（%）	CV
HPA-1a（IU）
0.9	0.87	96.20	1.30	0.92	102.60	5.60
1.8	1.72	95.70	2.40	1.76	97.90	4.00
3.6	3.71	103.10	3.60	3.54	98.30	4.80
HPA-3a（AU）
3	3.07	102.20	5.10	3.02	100.80	5.90
10	9.79	97.90	2.70	10.09	100.30	5.50
15	14.38	103.10	5.20	15.20	101.30	4.40
HPA-5b（AU）
3	3.18	106.10	4.70	2.95	98.50	6.30
10	10.01	100.20	4.50	10.24	102.40	3.80
15	15.70	104.50	5.30	15.29	101.90	3.20