Vendramin Chiara, Thomas Mari, Westwood John-Paul, Scully Marie
Department of Haematology, University College London Hospital, London, United Kingdom.
Department of Haematology, University College London Hospital, Cardiometabolic Programme NIHR UCLH/UCL BRC, London, United Kingdom.
TH Open. 2018 Sep 26;2(3):e329-e333. doi: 10.1055/s-0038-1672187. eCollection 2018 Jul.
A diagnosis of thrombotic thrombocytopenic purpura (TTP) is confirmed by a severe deficiency (<10%) of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity. Autoantibodies to ADAMTS13 can be detected with a simplified enzyme-linked immunosorbent assay (ELISA). An alternative methodology is a Bethesda assay, which has never been formally assessed in TTP. This study aimed to investigate the inhibitory anti-ADAMTS13 antibody assay and determine if the Bethesda assay is advantageous compared with the ELISA, measuring total immunoglobulin G (IgG) antibodies to ADAMTS 13. The Bethesda method determines the neutralizing activity of anti-ADAMTS13 antibodies in pooled normal plasma. We selected six immune-mediated TTP (iTTP) patients with ADAMTS13 activity levels <10% and strong ADAMTS13 inhibitors by 50:50 mixing studies and analyzed anti-ADAMTS13 antibodies using the Bethesda and ELISA assays. ADAMTS13 activity was stable at room temperature, while a time-dependent decrease in activity was detected in assay conditions of 37°C. Adding 5 mM Ca to citrated plasma prevented loss of ADAMTS13 activity with time. There was time dependence to the antibody-mediated inactivation, after 2-hour incubation. Two of the iTTP patients had no detectable ADAMTS13 antibodies by the Bethesda assay, but had high titer of anti-ADAMTS13 antibodies and low ADAMTS13 antigen levels. The Bethesda assay can only detect anti-ADAMTS13 antibodies that functionally inhibit ADAMTS13. The anti-ADAMTS13 IgG ELISA instead allows the rapid identification of total IgG autoantibodies, detecting both inhibitory and noninhibitory antibodies.
通过具有血小板反应蛋白-1型基序的解聚素和金属蛋白酶13（ADAMTS13）活性严重缺乏（<10%）来确诊血栓性血小板减少性紫癜（TTP）。可通过简化的酶联免疫吸附测定（ELISA）检测针对ADAMTS13的自身抗体。另一种方法是贝塞斯达测定法，该方法从未在TTP中进行过正式评估。本研究旨在研究抑制性抗ADAMTS13抗体测定法，并确定与ELISA相比，贝塞斯达测定法在测量针对ADAMTS 13的总免疫球蛋白G（IgG）抗体方面是否具有优势。贝塞斯达方法可确定抗ADAMTS13抗体在混合正常血浆中的中和活性。我们通过50:50混合研究选择了6例ADAMTS13活性水平<10%且具有强ADAMTS13抑制剂的免疫介导性TTP（iTTP）患者，并使用贝塞斯达测定法和ELISA测定法分析抗ADAMTS13抗体。ADAMTS13活性在室温下稳定，而在37°C的测定条件下检测到活性随时间下降。向枸橼酸血浆中添加5 mM Ca可防止ADAMTS13活性随时间丧失。孵育2小时后，抗体介导的失活存在时间依赖性。2例iTTP患者通过贝塞斯达测定法未检测到可检测的ADAMTS13抗体，但具有高滴度的抗ADAMTS13抗体和低ADAMTS13抗原水平。贝塞斯达测定法只能检测功能上抑制ADAMTS13的抗ADAMTS13抗体。相反，抗ADAMTS13 IgG ELISA可快速鉴定总IgG自身抗体，检测抑制性和非抑制性抗体。
