Remacle Albert G, Dolkas Jennifer, Angert Mila, Hullugundi Swathi K, Chernov Andrei V, Jones R Carter W, Shubayev Veronica I, Strongin Alex Y
Infectious and Inflammatory Disease Center/Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
Department of Anesthesiology, University of California, San Diego, La Jolla, CA 92093, USA; VA San Diego Healthcare System, La Jolla, CA 92037, USA.
J Immunol Methods. 2018 Apr;455:80-87. doi: 10.1016/j.jim.2018.02.002. Epub 2018 Feb 8.
Sciatic nerve chronic constriction injury (CCI) in rodents produces nerve demyelination via proteolysis of myelin basic protein (MBP), the major component of myelin sheath. Proteolysis releases the cryptic MBP epitope, a demyelination marker, which is hidden in the native MBP fold. It has never been established if the proteolytic release of this cryptic MBP autoantigen stimulates the post-injury increase in the respective circulating autoantibodies. To measure these autoantibodies, we developed the ELISA that employed the cryptic 84-104 MBP sequence (MBP84-104) as bait. This allowed us, for the first time, to quantify the circulating anti-MBP84-104 autoantibodies in rat serum post-CCI. The circulating IgM (but not IgG) autoantibodies were detectable as soon as day 7 post-CCI. The IgM autoantibody level continually increased between days 7 and 28 post-injury. Using the rat serum samples, we established that the ELISA intra-assay (precision) and inter-assay (repeatability) variability parameters were 2.87% and 4.58%, respectively. We also demonstrated the ELISA specificity by recording the autoantibodies to the liberated MBP84-104 epitope alone, but not to intact MBP in which the 84-104 region is hidden. Because the 84-104 sequence is conserved among mammals, we tested if the ELISA was applicable to detect demyelination and quantify the respective autoantibodies in humans. Our limited pilot study that involved 16 female multiple sclerosis and fibromyalgia syndrome patients demonstrated that the ELISA was efficient in measuring both the circulating IgG- and IgM-type autoantibodies in patients exhibiting demyelination. We believe that the ELISA measurements of the circulating autoantibodies against the pathogenic MBP84-104 peptide may facilitate the identification of demyelination in both experimental and clinical settings. In clinic, these measurements may assist neurologists to recognize patients with painful neuropathy and demyelinating diseases, and as a result, to personalize their treatment regimens.
啮齿动物的坐骨神经慢性压迫性损伤（CCI）通过髓鞘碱性蛋白（MBP，髓鞘的主要成分）的蛋白水解作用导致神经脱髓鞘。蛋白水解作用释放出隐藏在天然MBP折叠结构中的隐蔽MBP表位，这是一种脱髓鞘标志物。这种隐蔽MBP自身抗原的蛋白水解释放是否会刺激损伤后相应循环自身抗体的增加，目前尚未得到证实。为了检测这些自身抗体，我们开发了一种酶联免疫吸附测定（ELISA），该方法使用隐蔽的84 - 104 MBP序列（MBP84 - 104）作为诱饵。这使我们首次能够定量CCI后大鼠血清中循环的抗MBP84 - 104自身抗体。CCI后第7天即可检测到循环IgM（而非IgG）自身抗体。损伤后第7天至28天，IgM自身抗体水平持续升高。使用大鼠血清样本，我们确定ELISA测定内（精密度）和测定间（重复性）变异参数分别为2.87%和4.58%。我们还通过记录仅针对释放的MBP84 - 104表位而非84 - 104区域隐藏的完整MBP的自身抗体，证明了ELISA的特异性。由于84 - 104序列在哺乳动物中是保守的，我们测试了该ELISA是否适用于检测人类的脱髓鞘并定量相应的自身抗体。我们涉及16名女性多发性硬化症和纤维肌痛综合征患者的有限初步研究表明，该ELISA在测量表现出脱髓鞘的患者循环IgG型和IgM型自身抗体方面是有效的。我们认为，针对致病性MBP84 - 104肽的循环自身抗体的ELISA测量可能有助于在实验和临床环境中识别脱髓鞘。在临床上，这些测量可能有助于神经科医生识别患有疼痛性神经病变和脱髓鞘疾病的患者，从而使他们能够个性化治疗方案。
