Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti-idiotype monoclonal antibodies
Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti-idiotype monoclonal antibodies
复制标题
DOI：
10.1111/jth.14135
复制
发表时间：
2018-07-01
影响因子：
10.4
通讯作者：
Shima, M.
中科院分区：
文献类型：
--
作者：
Nogami, K.;Soeda, T.;Shima, M.
背景：Emicizumab是一种抗因子（F）IXa/X双特异性单克隆抗体（mAb），模拟因子（F）VIIIa辅因子活性。Emicizumab不需要凝血酶活化，其对活化部分凝血酶原时间（APTT）的缩短作用比因子（F）VIII更明显。基于APTT的FVIII活性（FVIII：C）和FVIII抑制剂滴度测量受emicizumab存在的影响。目的：建立一种可靠的基于APTT的测定法，用于在emicizumab存在下测量FVIII。方法：使用一阶段F VIII：C和Bethesda抑制剂测定法研究了不含或含抑制剂的血友病A（HA）患者的血浆。制备了两种针对emicizumab的重组抗独特型mAb（抗emicizumab mAb），rcAQ 8针对抗FIXa-Fab，rcAJ 540针对抗FX-Fab。结果如下：联合抗独特型mAb（各2000 nm）通过竞争性抑制抗体与FIX（a）/FX（a）的结合，消除了emicizumab对HA血浆APTT（不含或含抑制剂）的影响。在所有参考水平的FVIII下，在存在emicizumab（1 μ m =类似于150 μ g mL（-1））的情况下，对无抑制剂的HA血浆中FVIII凝血活性的测量被高估。向试验混合物中加入抗emicizumab mAb可完全中和emicizumab，有助于准确测定FVIII：C。在含有抑制剂的HA血浆或与抗FVIII中和抗体混合的正常血浆中存在emicizumab时，无法检测到抗FVIII抑制剂滴度。emicizumab的这些作用通过添加抗独特型mAb完全抵消，从而可以准确评估抑制剂滴度。结论：在标准一期凝血试验中体外纳入抗emicizumab mAb可防止emicizumab干扰，并可准确测量FVIII：C和抑制剂滴度。
Background: Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim: To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods: Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results: The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 mu m = similar to 150 mu g mL(-1)) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion: The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.