Morgenthaler Nils G, Minich Waldemar B, Willnich Marita, Bogusch Thomas, Hollidt Jörg M, Weglöhner Wolfgang, Lenzner Cornelia, Bergmann Andreas
Research Department of BRAHMS AG, Biotechnology Center Hennigsdorf/Berlin, Neuendorfstr. 25, D-16761 Hennigsdorf, Germany.
Mol Cell Endocrinol. 2003 Dec 30;212(1-2):73-9. doi: 10.1016/j.mce.2003.09.018.
Purification of TSH receptor autoantibodies (TRAb) from the serum of patients with Graves' disease (GD) might help to elucidate the nature of these disease causing autoantibodies. We describe here for the first time the successful affinity purification of human TRAb. Affinity purification was performed in a four step procedure with human recombinant TSH receptor (TSH-R) expressed in K562 cells. Purification from six different serum pools from patients with GD and two individual sera (one with only thyroid stimulating antibodies (TSAb) one with only thyroid blocking antibodies (TBAb)) resulted in a purity of 39.2+/-3.8 IU/mg TRAb or 25.7+/-2.1 microg IgG/IU (about 3.5-13.7 microg TRAb/ml serum). The average enrichment based on the respective original serum was 3420-fold (range 1200-10,000). The kDa of the purified TRAb were in the range of 0.7-2.6 x 10(-10)M. All purified TRAb (except from the TBAb serum which showed blocking activity) showed a more than 1000-fold stronger stimulation in the TSAb bioassay based on the IgG content than the original serum, and similar stimulation based on international units (IU/l) TRAb. When labelled purified TRAb were used in a competitive assay as tracer instead of bovine TSH, their binding to the human recombinant TSH-R on tubes was displaced by 99 of 100 GD sera (selected for TBII activity). Correlation to the standard TSH tracer was r=0.92. Interestingly, the use of TRAb tracer derived from a patient with TSAb and a patient with TBAb gave virtually identical results (r=0.93) with these patients, suggesting similar if not identical binding sites for both TRAb subtypes. In conclusion, this is the first report on the purification of human TRAb from the serum of patients with GD. The purified TRAb are of low concentration with high affinity, strong TBII and TSAb activity. Further characterisation may allow new insights in TRAb epitope localisation, the pathology of GD and the differences between TSAb and TBAb. Also, their use as tracer in a competitive assay is the first report on a completely homogenous assay with high sensitivity for TSH-R autoantibodies.
从格雷夫斯病（GD）患者血清中纯化促甲状腺激素受体自身抗体（TRAb）可能有助于阐明这些致病自身抗体的性质。我们在此首次描述了人TRAb的成功亲和纯化。亲和纯化采用四步法，使用在K562细胞中表达的人重组促甲状腺激素受体（TSH-R）。从6个不同的GD患者血清池和2份个体血清（1份仅含甲状腺刺激抗体（TSAb），1份仅含甲状腺阻断抗体（TBAb））中进行纯化，得到的TRAb纯度为39.2±3.8 IU/mg，或25.7±2.1 μg IgG/IU（约3.5 - 13.7 μg TRAb/ml血清）。基于各自原始血清的平均富集倍数为3420倍（范围为1200 - 10000）。纯化后的TRAb的kDa在0.7 - 2.6×10⁻¹⁰M范围内。所有纯化的TRAb（除显示阻断活性的TBAb血清外）基于IgG含量在TSAb生物测定中的刺激作用比原始血清强1000倍以上，基于国际单位（IU/l）TRAb的刺激作用相似。当用标记的纯化TRAb作为示踪剂用于竞争测定而非牛TSH时，1OO份GD血清（因促甲状腺激素受体抗体抑制试验（TBII）活性而选择）中有99份能使其与管上的人重组TSH-R的结合被取代。与标准TSH示踪剂的相关性为r = 0.92。有趣的是，使用来自TSAb患者和TBAb患者的TRAb示踪剂对这些患者进行检测，结果几乎相同（r = 0.93），这表明两种TRAb亚型的结合位点相似甚至相同。总之，这是关于从GD患者血清中纯化人TRAb的首次报道。纯化后的TRAb浓度低、亲和力高、TBII和TSAb活性强。进一步的特性分析可能有助于深入了解TRAb表位定位、GD的病理学以及TSAb和TBAb之间的差异。此外，将其用作竞争测定中的示踪剂是关于TSH-R自身抗体高灵敏度完全同质测定的首次报道。
