Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.
Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.
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DOI：
10.1172/jci111187
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发表时间：
1984
期刊：
影响因子：
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通讯作者：
Robert;D. Schreiber;Neil;-R.;Cooper
中科院分区：
文献类型：
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作者：
Robert;D. Schreiber;Neil;-R.;Cooper
我们开发了一种新的、特异的、高灵敏的酶联免疫吸附试验(ELISA)，它可以定量检测人血清、血浆或激活剂表面的替代途径的激活。ELISA检测补体的第三组分(C3b)、补体因子B(BB)的蛋白分解片段和备解素(P)复合体或其衍生产物C3b，P。在该方法中，将激活剂-血浆混合物、含有激活替代途径的血浆或其他样品加入预涂有P抗体的微量滴定板孔中，通过随后添加酶标记的抗C3来定量结合的C3b、BB、P或C3b，P复合体。通过参照C3标准曲线，所产生的显色底物的水解被表示为毫微克的C3b。由于检测到的复合体的性质，该方法除了具有激活该途径的绝对特异性外，还具有很高的灵敏度，能够重复性地检测血清中10-20 ng/ml的C3b，P复合体。这个值相当于血清中C3的0.0015%。在一系列验证酶联免疫吸附试验参数的研究中，发现反应性取决于替代途径蛋白的存在、途径的功能完整性和镁的存在。用神经氨酸酶处理绵羊红细胞，使其转化为激活剂。通过使用多种激活剂，用ELISA法测定的活化动力学和结合的C3b分子数与用C3b沉淀法测定的结果非常相似。当以镁EGTA血清和纯化的替代途径蛋白的混合物作为途径的来源时，ELISA也检测到相同的激活动力学。ELISA反应动力学也与限制指数相关，限制指数是衡量替代途径激活能力的指标。这些研究累积验证了酶联免疫吸附试验作为替代途径激活的直接和定量检测。这种酶联免疫吸附试验的敏感性使得它可以用来检测几种病毒对直接替代途径的激活。酶联免疫吸附试验还表明，某些经典途径激活剂触发了替代途径的扩增环，而其他途径则不会。此外，在血清与某些激活剂的混合物的上清液中出现了稳定的ELISA反应复合体，而不是其他激活剂。ELISA检测已经发生的激活的能力和反应复合体的稳定性使临床血清研究成为可能。正常人血清(20份)中含有较低水平(5-20 ng/ml)的ELISA-反应性复合体。成人呼吸窘迫综合征(9-10)、伤寒(8-10)、疟疾(3-5)、革兰氏阴性败血症(9/47)、急性创伤和休克(6/25)和系统性红斑狼疮(3/29)患者的血清在另一种途径的ELISA法中显示出高水平的复合体反应。相比之下，循环C3肾病因子患者的9份血清在酶联免疫吸附试验中均为阴性。
We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA.