Mörtberg Anette, Meinke Stephan, Berg Petra, Killie Mette Kjær, Kjeldsen-Kragh Jens, Järås Kerstin, Refsum Erle, Höglund Petter, Wikman Agneta
Dept. of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden; Dept. of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.
Dept. of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden; Dept. of Medicine Huddinge, Center for Hematology and Regenerative Medicine (HERM), Stockholm, Sweden.
J Immunol Methods. 2016 Jul;434:9-15. doi: 10.1016/j.jim.2016.04.001. Epub 2016 Apr 5.
We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT).
我们开发了一种改良的单克隆抗体固定化血小板抗原检测法（MAIPA），通过使用生物素化单克隆抗体、链霉亲和素包被的磁珠以及流式细胞术检测，提高了检测抗人血小板抗原（HPA）抗体的灵敏度。与内部标准MAIPA相比，磁珠-MAIPA在检测抗HPA-1a和抗HPA-5b时具有更高的信噪比分辨率（高出10倍以上）。此外，还展示了对抗HPA-15（CD109）的高效且可重复的检测。使用世界卫生组织抗HPA-1a、抗HPA-3a和抗HPA-5b国际参考试剂证实了灵敏度的提高，这使得能够与其他实验室比较检测终点。最后，对磁珠-MAIPA进行了抗HPA-1a定量验证。磁珠-MAIPA的定量下限为0.4IU/mL，而之前报道的标准MAIPA为1IU/mL。基于对所有测试的HPA抗体的性能改进，磁珠-MAIPA已在我们实验室中取代标准MAIPA，用于诊断因HPA免疫引起的疾病，如胎儿和新生儿同种免疫性血小板减少症（FNAIT）。
