Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, GR 11521, Athens, Greece.
J Neuroimmunol. 2011 Dec 15;240-241:79-86. doi: 10.1016/j.jneuroim.2011.09.007. Epub 2011 Oct 10.
Autoimmune myasthenia gravis is usually characterized by the presence of autoantibodies against the acetylcholine receptor (80-90% of patients) or muscle-specific tyrosine kinase (MuSK) (5% of patients). In the remaining patients, no such antibodies (Abs) are detectable, but this could be due either to the presence of auto-Abs to yet unidentified antigens or to concentrations of circulating Abs below the threshold of the available assays. The most popular and sensitive assay for anti-MuSK Abs is a radioimmunoprecipitation assay (RIPA), which uses (125)I-labeled MuSK as test antigen. A serious limiting factor of the sensitivity of such RIPAs is that small volumes of test serum are required (maximum 5-20 μl) in order to avoid excessive background values. We have overcome this obstacle by the development of a two-step RIPA. This involves non-stringent affinity purification of anti-MuSK Abs from a large volume of patient's serum, followed by a regular RIPA step, with the isolated Abs, to determine the antibody titer. This two-step assay was shown to be 10-50 times more sensitive than the regular RIPA. All tested sera previously found to be positive in the regular RIPA and normal sera were also positive or negative in the two-step RIPA, respectively. Importantly, of seven tested sera previously characterized by regular RIPA as negative with titers that were above zero, but statistically not significantly higher from the background, two were found to be positive, while the others were clearly shown to be negative. We conclude that our diagnostic test can detect very low concentrations of circulating anti-MuSK Abs. The general principle of this two-step RIPA approach may also be applied to the detection of currently undetectable concentrations of circulating auto-Abs involved in other diseases.
自身免疫性重症肌无力通常表现为存在针对乙酰胆碱受体的自身抗体（80-90%的患者）或肌肉特异性酪氨酸激酶（MuSK）（5%的患者）。在其余患者中，无法检测到此类抗体（Abs），但这可能是由于存在针对尚未识别抗原的自身-Abs 或由于循环 Abs 浓度低于现有检测方法的阈值。用于检测抗 MuSK Abs 的最流行和敏感的检测方法是放射性免疫沉淀测定法（RIPA），该方法使用（125）I 标记的 MuSK 作为测试抗原。此类 RIPA 的灵敏度的一个严重限制因素是需要使用小体积的测试血清（最大 5-20 μl），以避免背景值过高。我们通过开发两步 RIPA 克服了这一障碍。这涉及从大量患者血清中非严格亲和纯化抗 MuSK Abs，然后进行常规 RIPA 步骤，用分离的 Abs 来确定抗体滴度。与常规 RIPA 相比，两步法检测的灵敏度提高了 10-50 倍。所有以前在常规 RIPA 中检测为阳性的测试血清和正常血清在两步 RIPA 中也分别为阳性或阴性。重要的是，在以前用常规 RIPA 检测为阴性且滴度高于零但统计学上不显著高于背景的七种测试血清中，有两种被检测为阳性，而其他则被明确检测为阴性。我们得出结论，我们的诊断测试可以检测到非常低浓度的循环抗 MuSK Abs。这种两步 RIPA 方法的一般原理也可应用于检测目前无法检测到的涉及其他疾病的循环自身 Abs 浓度。
