Hellmark T, Brunmark C, Trojnar J, Wieslander J
Department of Nephrology, Lund University, Sweden.
Clin Exp Immunol. 1996 Sep;105(3):504-10. doi: 10.1046/j.1365-2249.1996.119808.x.
Autoantibodies to the non-collagenous (NC1) domain of the alpha 3(IV)-chain of type IV collagen are found in sera from patients with anti-GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture's syndrome and from a patient with atypical anti-GBM antibodies, recognizing the alpha 1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the alpha 1(IV)- and alpha 3(IV)-chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti-GBM antibodies recognized a 20 amino acid peptide from the alpha 1(IV)-chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C-terminal amino acids of the alpha 1(IV)-chain, with the sequence 1754-MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native alpha 1(IV)-chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti-GBM antibodies, since the patient with anti-alpha 1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-alpha 3(IV) antibodies had a rapidly progressive disease.
在抗肾小球基底膜（GBM）肾炎患者的血清中发现了针对IV型胶原α3（IV）链非胶原（NC1）结构域的自身抗体。这些抗体已被证明具有致病性。在本研究中，对患有Goodpasture综合征的患者以及一名仅识别α1（IV）链的非典型抗GBM抗体患者的抗体特异性进行了研究。在夹心ELISA和竞争ELISA中使用了覆盖α1（IV）链和α3（IV）链完整NC1结构域的重叠合成肽。没有一份Goodpasture综合征患者的血清对合成肽有反应。然而，来自非典型抗GBM抗体患者的抗体识别出α1（IV）链上的一个20个氨基酸的肽段。通过对不同氨基酸进行甘氨酸替代，进一步缩小了反应性肽段的范围。我们已将表位定位到α1（IV）链C末端的最后四个氨基酸，序列为1754-MRRT。发现两个精氨酸残基对于抗体结合至关重要。苏氨酸很重要，而甲硫氨酸的重要性较低。这四个氨基酸也被确定为能够抑制自身抗体与天然α1（IV）链结合的最小肽段。本研究表明重叠肽可用于绘制线性表位。然而，对于构象表位，如Goodpasture表位，则必须使用其他方法。了解抗GBM抗体的精细特异性在预后方面很重要，因为患有抗α1（IV）抗体的患者病情较轻，而患有抗α3（IV）抗体的Goodpasture综合征患者病情进展迅速。
