Zilow G, Naser W, Rutz R, Burger R
Institute for Immunology, University of Heidelberg, F.R.G.
J Immunol Methods. 1989 Jul 26;121(2):261-8. doi: 10.1016/0022-1759(89)90169-5.
C3a levels in plasma are usually measured by a competitive inhibition radioimmunoassay (RIA) using 125I-labelled C3a-desArg and antibodies to C3a capable of detecting C3a determinants which are also present on the native C3. Therefore, prior to the assay native, non-cleaved C3 has to be removed completely from the C3a-containing sample by precipitation. We developed a new rapid two-site sandwich ELISA system for the quantitation of C3a-desArg in plasma. This immunoassay uses a monoclonal antibody (mAb H466) reacting with C3a-desArg but not with C3. The reactivity of mAb H466 with a neoantigenic determinant of C3a-desArg permitted the direct quantitation of C3a-desArg without removal of C3 from the sample. The mAb H466 was used as a capture antibody and bound C3a-desArg was detected with a second peroxidase-labelled anti-C3a mAb. The lower limit of detection of C3a-desArg in this ELISA was 1 ng/ml. The C3a-desArg levels measured in the plasma samples of various patients were found to differ over a wide range. A good correlation was observed between the results obtained in the RIA and those obtained in the ELISA (r = 0.95). High levels of C3a-desArg were detected in plasma from patients with multiple trauma and patients undergoing haemodialysis. The C3a-desArg assay described should facilitate the routine quantitation of C3a in samples of plasma.
血浆中C3a水平通常采用竞争性抑制放射免疫测定法（RIA）进行检测，该方法使用125I标记的C3a-去精氨酸以及能够检测天然C3上也存在的C3a决定簇的抗C3a抗体。因此，在检测之前，必须通过沉淀从含C3a的样品中完全去除天然的、未裂解的C3。我们开发了一种新的快速双位点夹心ELISA系统，用于定量血浆中的C3a-去精氨酸。这种免疫测定法使用一种单克隆抗体（mAb H466），它与C3a-去精氨酸反应，但不与C3反应。mAb H466与C3a-去精氨酸的新抗原决定簇的反应性使得无需从样品中去除C3即可直接定量C3a-去精氨酸。mAb H466用作捕获抗体，结合的C3a-去精氨酸用第二种过氧化物酶标记的抗C3a单克隆抗体进行检测。该ELISA中C3a-去精氨酸的检测下限为1 ng/ml。在各种患者的血浆样本中测得的C3a-去精氨酸水平差异很大。在RIA和ELISA中获得的结果之间观察到良好的相关性（r = 0.95）。在多发伤患者和接受血液透析的患者的血浆中检测到高水平的C3a-去精氨酸。所述的C3a-去精氨酸检测方法应有助于血浆样本中C3a的常规定量。
