Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China.
Nephrology (Carlton). 2012 Feb;17(2):160-6. doi: 10.1111/j.1440-1797.2011.01511.x.
Cases with anti-glomerular basement membrane (GBM) disease have been reported with linear deposit of immunoglobulin G (IgG) along GBM, but have undetectable anti-GBM antibodies in circulation by enzyme linked immunosorbent assays (ELISA). We speculated that the structure of the antigens recognized by these antibodies may contribute to the negative results of ELISA.
Sera from four patients were collected, with typical linear deposit of IgG along GBM but no anti-GBM reactivity by commercial ELISA kits. Circulating anti-GBM antibodies were detected by indirect immunofluorescence. Antigen specificity and its conformational structure was investigated by western-blot analysis, using recombinant human α1-α5(IV)NC1 and chimeric proteins E(A) and E(B) as antigens.
The presence of circulating anti-GBM antibodies were confirmed by indirect immunofluorescence with linear deposit of IgG towards cryptic epitopes along GBM on normal kidney sections. These antibodies did not recognize recombinant human α1, α2, α4 or α5(IV)NC1, but could blot α3(IV)NC1 under non-reducing non-boiling conditions on western-blot analysis, when the conformational epitope(s) on α3(IV)NC1 were thought to be preserved. When α3(IV)NC1 was prepared under reducing conditions with β-mercaptoethanol and/or boiled to destroy the disulfide bonds, the binding with the antibodies disappeared. Moreover, these antibodies recognized neither E(A) nor E(B) , indicating their distinct epitope repertoire.
Circulating anti-GBM antibodies undetectable by ELISA could recognize cryptic and conformation-dependent epitopes restricted on α3(IV)NC1, distinct from E(A) and E(B) . Indirect immunofluorescence was necessary for antibody detection and treatment monitoring under such circumstances.
已有研究报道，抗肾小球基底膜（GBM）病患者的血清中可检测到沿 GBM 线性沉积的免疫球蛋白 G（IgG），但通过酶联免疫吸附试验（ELISA）检测不到循环中的抗 GBM 抗体。我们推测这些抗体所识别的抗原结构可能导致 ELISA 检测结果为阴性。
收集了 4 例具有典型 GBM 线性 IgG 沉积但商业 ELISA 试剂盒无抗 GBM 反应性的患者血清。通过间接免疫荧光法检测循环抗 GBM 抗体。使用重组人α1-α5（IV）NC1 和嵌合蛋白 E（A）和 E（B）作为抗原，通过 Western-blot 分析检测抗原特异性及其构象结构。
通过间接免疫荧光法在正常肾脏切片上检测到线性沉积的 IgG，证实了循环抗 GBM 抗体的存在，该 IgG 针对 GBM 上的隐匿表位。这些抗体不能识别重组人α1、α2、α4 或α5（IV）NC1，但在 Western-blot 分析中可以在非还原非煮沸条件下检测到α3（IV）NC1，此时认为α3（IV）NC1 上的构象表位得以保留。当用β-巯基乙醇还原并/或煮沸破坏二硫键时，α3（IV）NC1 制备条件还原，与抗体的结合消失。此外，这些抗体既不识别 E（A）也不识别 E（B），表明其具有独特的表位谱。
ELISA 检测不到的循环抗 GBM 抗体可识别局限于α3（IV）NC1 的隐匿和构象依赖性表位，与 E（A）和 E（B）不同。在这种情况下，需要进行间接免疫荧光法来进行抗体检测和治疗监测。
