Fischer Christian, Buschmann Katharina, Blank Miri, Stachl Angelika, Miesbach Wolfgang, Shoenfeld Yehuda, Lackner Karl J, von Landenberg Philipp
Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University, Mainz, Germany.
Immunobiology. 2006;211(9):695-9. doi: 10.1016/j.imbio.2006.08.001. Epub 2006 Sep 7.
Our objective was to characterize monoclonal antiphospholipid antibodies (APL) and identify disease-associated antigens in patients with the antiphospholipid syndrome (APS). We used the monoclonal antibody HL-5B, derived from a patient with APS suffering from multiple ischemic events, to screen a 12-mer peptide phage display library (New England Biolabs, London, England). The identified phage clones were sequenced and the derived consensus peptide was synthesized. The peptide was used to perform competitive inhibition experiments for their ability to inhibit the binding of the monoclonal antibody and of serum antibodies to cardiolipin and phosphatidylserine. Additionally patients and control sera were screened for their binding reactivities to this peptide. Using this 12-mer phage display library the peptide APHKHKASLSIY as consensus peptide for the monoclonal antiphospholipid antibody HL-5B could be identified. In competitive inhibition studies we showed that this peptide is able to inhibit the binding of HL-5B to cardiolipin and phosphatidylserine and furthermore another antiphospholipid antibody used as control was also inhibited in its binding to phospholipids. Using 21 sera from APS patients 67% showed a binding to the peptide in a specific ELISA above the cutoff level, generated with sera from 20 healthy controls. Out of the reactive patients' sera we used two exemplarily to perform inhibition studies. Both sera could be inhibited more than 40% in their binding to cardiolipin in a commercially available antiphospholipid antibody assay (Aescu.diagnostics, Wendelsheim, Germany). The identified peptide APHKHKASLSIY simulates the antigenic structure recognized from a subpopulation of serum antiphospholipid antibodies. This might indicate that the diversity of the antiphospholipid antibodies is limited and only few epitopes or few common structures are responsible for the development of those antibodies. Tests using these epitopes will strongly improve laboratory diagnosis of the APS.
我们的目标是对单克隆抗磷脂抗体（APL）进行表征，并鉴定抗磷脂综合征（APS）患者中与疾病相关的抗原。我们使用了源自一名患有多次缺血事件的APS患者的单克隆抗体HL-5B，来筛选一个12肽噬菌体展示文库（新英格兰生物实验室，英国伦敦）。对鉴定出的噬菌体克隆进行测序，并合成推导的共有肽。该肽用于进行竞争抑制实验，以检测其抑制单克隆抗体以及血清抗体与心磷脂和磷脂酰丝氨酸结合的能力。此外，还检测了患者和对照血清与该肽的结合反应性。利用这个12肽噬菌体展示文库，可鉴定出作为单克隆抗磷脂抗体HL-5B共有肽的APHKHKASLSIY肽。在竞争抑制研究中，我们发现该肽能够抑制HL-5B与心磷脂和磷脂酰丝氨酸的结合，此外，用作对照的另一种抗磷脂抗体与磷脂的结合也受到抑制。使用21份APS患者的血清，在特异性酶联免疫吸附测定（ELISA）中，67%的血清与该肽的结合高于由20名健康对照者血清确定的临界值。从有反应的患者血清中，我们示例性地选取了两份进行抑制研究。在市售的抗磷脂抗体检测（德国温德尔斯海姆的爱思试剂诊断公司）中，这两份血清与心磷脂的结合均能被抑制40%以上。鉴定出的APHKHKASLSIY肽模拟了血清抗磷脂抗体亚群识别的抗原结构。这可能表明抗磷脂抗体的多样性有限，只有少数表位或少数共同结构导致了这些抗体的产生。使用这些表位进行检测将大大改善APS的实验室诊断。
