Arroyave C M, Tan E M
J Immunol Methods. 1976;13(2):101-12. doi: 10.1016/0022-1759(76)90148-4.
Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by asssaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.
对流免疫电泳（CIE）被用作检测补体系统第三成分（C3）激活的方法。高度纯化的C3、正常人血清（NHS）、乙二胺四乙酸（EDTA）处理的血浆以及用聚合人免疫球蛋白（agg-IgG）或菊粉激活的血清被用作C3和/或C3裂解产物的来源。在乙二醇双四乙酸（EGTA，10 mM）和氯化镁（MgCl2，0.3 mM）存在的条件下测定补体替代途径的激活，这些条件可阻断经典途径的激活。当在CIE中用针对整个C3或C3裂解产物的抗血清检测纯化的天然C3、新鲜NHS和新鲜EDTA血浆时，仅发现一条沉淀线，其被鉴定为天然C3。然而，当用agg-IgG或菊粉激活的血清针对相同试剂进行检测时，可见两条沉淀线。第一条沉淀线的阴极迁移率更高，与天然C3的沉淀线相同。第二条沉淀线的阳极迁移率更高，与第一条明显分开，并且如用特异性抗血清进行免疫化学检测所示，其含有C3c和C3d。通过这种CIE方法，在显示总补体（CH50）水平低于正常而有补体激活证据的患者中鉴定出天然C3和C3裂解产物。当鉴定出C3裂解产物时，即使血清中C3浓度在0.25 mg/ml至1.5 mg/ml之间变化，C3c-C3d沉淀线也总能因其不同的电泳迁移率而与天然C3区分开来。通过在将agg-IgG添加到新鲜NHS后的不同时间间隔进行测定，将CIE的灵敏度与CH50的灵敏度进行了比较。在CH50出现任何下降之前以及在CH50显著降低的所有时间，CIE均检测到了C3c-C3d裂解产物。本研究表明，CIE技术是一种用于检测人类疾病中通过经典或替代途径激活补体系统的高度灵敏、特异且快速的方法。
