Rossen Marie, Iversen Kasper, Teisner Ane, Teisner Børge, Kliem Anette, Grudzinskas Gedis
Immunology and Microbiology, University of Southern Denmark, Odense, Denmark.
Clin Biochem. 2007 Apr;40(7):478-84. doi: 10.1016/j.clinbiochem.2006.11.025. Epub 2007 Jan 18.
PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients with acute coronary syndrome (ACS). The aims of the present study were to demonstrate (i) the importance of antibody specificity, (ii) the potential pitfalls in changing assay technology, (iii) the importance of strict definition of technology, and (iv) the application of a well-defined assay technology on sera from patients with ACS.
Candidate monoclonal antibodies (Mab) were identified by immunohistochemistry, Western blot and the absence of positive signals (ELISA) with normal, non-pregnant serum as antigen source. The ELISA technology was standardized against the original PAPP-A RIA and the WHO reference preparation (WHO 78/610). Results different from those obtained by the original RIA led to ELISA modifications with respect to dilution buffer and enzymatic digestion of the Mab.
The first generation ELISA revealed serum measurements from a pool of non-pregnant (n=103) individuals which, compared to the RIA, seemed to be false positive. The false positive reaction was abolished by addition of bovine serum (BS) to the dilution buffer. Subsequent analysis of individual sera (n=103) indicated that 7/103 were still false positive. This reaction was eliminated by introduction of F(ab')(2)-fragment of the indicator antibody. This modified ELISA revealed that serum PAPP-A levels in ACS were statistically significantly higher than in controls (p<0.001). Moreover, serum PAPP-A in ACS patients with ST-segment elevation (STEMI) were higher (p<0.001) compared to patients without ST-segment elevation (NSTEMI). Immunohistochemical analysis failed to identify PAPP-A in the atherosclerotic plaques.
妊娠相关血浆蛋白A（PAPP-A）已成为孕早期唐氏综合征筛查的主要生化血清标志物，原始数据基于放射免疫分析（RIA）结果。近期使用夹心酶联免疫吸附测定（ELISA）技术的观察结果表明，PAPP-A可能是急性冠状动脉综合征（ACS）患者的一个潜在标志物。本研究的目的是证明：（i）抗体特异性的重要性；（ii）改变检测技术时的潜在陷阱；（iii）严格定义技术的重要性；（iv）将一种定义明确的检测技术应用于ACS患者的血清检测。
通过免疫组织化学、蛋白质印迹法以及以正常非妊娠血清作为抗原来源时无阳性信号（ELISA）来鉴定候选单克隆抗体（Mab）。ELISA技术依据原始的PAPP-A RIA和世界卫生组织参考制剂（WHO 78/610）进行标准化。与原始RIA获得的结果不同，这导致在稀释缓冲液和Mab的酶消化方面对ELISA进行了改进。
第一代ELISA显示，一组非妊娠个体（n = 103）的血清检测结果与RIA相比似乎为假阳性。通过在稀释缓冲液中添加牛血清（BS）消除了假阳性反应。随后对个体血清（n = 103）的分析表明，仍有7/103为假阳性。通过引入指示抗体的F(ab')(2)片段消除了该反应。这种改进后的ELISA显示，ACS患者的血清PAPP-A水平在统计学上显著高于对照组（p<0.001）。此外，与无ST段抬高的患者（NSTEMI）相比，ST段抬高型心肌梗死（STEMI）的ACS患者血清PAPP-A更高（p<0.001）。免疫组织化学分析未能在动脉粥样硬化斑块中鉴定出PAPP-A。
