Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique
Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique
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DOI：
10.1002/prca.201800157
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发表时间：
2019-07-01
影响因子：
2
通讯作者：
Antoine, Jean-Christophe
中科院分区：
文献类型：
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作者：
Moritz, Christian P.;Tholance, Yannick;Antoine, Jean-Christophe
目的鉴定血清自身抗体的自身抗原需要昂贵的方法，如蛋白质芯片或IP+MS，因此，通常通过免疫细胞化学/免疫组织化学的方法预先筛选血清中感兴趣的免疫表型。然而，区分免疫模式可能很困难，细胞内抗原也较难获得。因此，提出了一种简单、廉价的免疫印迹筛选方法，能够区分免疫模式和检测难治性蛋白。实验设计基于免疫印迹的自身抗原筛选的五个步骤：(1)蛋白质来源的选择，(2)蛋白质提取，(3)蛋白质分离，(4)蛋白质转移，(5)抗原检测。对52例慢性炎症性脱髓鞘性多发性神经病(CIDP)患者和45例正常对照血清进行筛查。结果蛋白来源对检测到的抗原集有影响。步骤2-4可适用于难降解蛋白质。此外，蛋白质带的纵向切割节省了75%的时间和材料，并允许准确地比较条带模式。由于后者是个体特异性和暂时恒定的，我们称它们为“免疫指纹”。在一项原则证明中，用两份抗神经束素-155阳性的CIDP血清和另外两条针对52名CIDP患者3-6亚群的免疫条带(120/220 kDa)检测到155 kDa的免疫条带。结论和临床意义适应性免疫印迹法是一种廉价、简便的准确筛选血清的方法，包括难治性抗原和细胞内抗原。
Purpose Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or IP+MS. Thus, sera are commonly pre-screened for interesting immunopatterns via immunocytochemistry/immunohistochemistry. However, distinguishing immunopatterns can be difficult and intracellular antigens are less accessible. Therefore, a simple and cheap immunoblot screening able to distinguish immunopatterns and to detect refractory proteins is presented. Experimental Design Five steps of immunoblotting-based autoantigen screening are revised: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, (5) antigen detection. Thereafter, 52 patients' sera with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls were screened. Results The protein source impacts the detected antigen set. Steps 2-4 can be adapted for refractory proteins. Furthermore, longitudinal cutting of protein lanes saves >= 75% of time and material and allows for exact comparison of band patterns. As the latter are individually specific and temporarily constant, we call them "immunological fingerprints". In a proof-of-principle, a 155 kDa immunoband was detected with two anti-neurofascin-155-positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3-6 of 52 CIDP patients. Conclusions and Clinical Relevance Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens.