Fornasieri A, Pinerolo C, Bernasconi P, Li M, Armelloni S, Gibelli A, D'Amico G
Division of Nephrology, San Carlo Borromeo Hospital, Milan, Italy.
Clin Nephrol. 1995 Aug;44(2):71-9.
In the present study we verified by solid phase ELISA the presence of antibodies against mesangial and endothelial cell constituents in patients with IgA-GN and Schoenlein-Henoch syndrome (SH). An antigen extract was prepared by sonication of human mesangial cell (MC) monolayers between third and fifth subculture and coated at 20 micrograms/ml on microtiter plates where sera were tested by incubation for 2 h at 37 degrees C and addition of peroxidase-conjugated anti-human IgG or IgA. In comparison to 86 normal controls, increased levels of IgG anti-MC antibodies were found in 15/84 patients with IgA-GN and 4/11 with SH. IgA antibodies were always negative. Furthermore anti-endothelial cell antibodies (AECA) were sought in the same patients and controls by ELISA as previously described. Increased levels of IgG and IgA AECA were found in 25/62 and 24/46 patients respectively. A cross-inhibition test showed that preadsorbment of positive sera for both IgG anti-MC and IgG AECA on endothelial cells in culture resulted in an inhibited binding of IgG to MC. HPLC-ELISA and Western blot analysis of the MC extract showed a significant binding of IgG from ELISA-positive sera to a protein band of 25-50 kD. Similar results were obtained by Western blot analysis of an endothelial cell extract. These results suggest the identity of the antigens recognized by IgG antibodies on endothelial cells and MC in patients with IgA-GN.
在本研究中，我们通过固相酶联免疫吸附测定法（ELISA）验证了IgA肾病（IgA-GN）和过敏性紫癜（SH）患者体内是否存在针对系膜细胞和内皮细胞成分的抗体。用人系膜细胞（MC）单层在第三代至第五代传代培养之间进行超声处理制备抗原提取物，并以20微克/毫升的浓度包被在微量滴定板上，将血清在37℃孵育2小时，然后加入过氧化物酶偶联的抗人IgG或IgA进行检测。与86名正常对照相比，在84例IgA-GN患者中有15例以及11例SH患者中有4例发现抗MC IgG抗体水平升高。IgA抗体始终为阴性。此外，通过ELISA法在相同的患者和对照中检测抗内皮细胞抗体（AECA），方法如前所述。分别在62例患者中的25例和46例患者中的24例发现IgG和IgA AECA水平升高。交叉抑制试验表明，将IgG抗MC和IgG AECA阳性血清预先吸附在培养的内皮细胞上，会导致IgG与MC的结合受到抑制。对MC提取物进行高效液相色谱-酶联免疫吸附测定（HPLC-ELISA）和蛋白质印迹分析显示，ELISA阳性血清中的IgG与一条25 - 50kD的蛋白条带存在显著结合。对内皮细胞提取物进行蛋白质印迹分析也得到了类似结果。这些结果表明，IgA-GN患者体内IgG抗体在内皮细胞和MC上识别的抗原具有同一性。
