Functional characterization of the lectin pathway of complement in human serum
Functional characterization of the lectin pathway of complement in human serum
复制标题
DOI：
10.1016/s0161-5890(02)00254-7
复制
发表时间：
2003-01-01
影响因子：
3.6
通讯作者：
Daha, MR
中科院分区：
文献类型：
--
作者：
Roos, A;Bouwman, LH;Daha, MR
甘露聚糖结合凝集素 (MBL) 是补体凝集素途径 (LP) 的主要引发剂。 MBL 基因外显子 I 的多态性与 MBL 功能受损和感染有关。评估血清中补体经典途径 (CP) 和旁路途径 (AP) 活性的功能测定广泛用于患者诊断。我们现在开发了一种功能性 LP 测定，能够对人血清中自体 MBL 依赖性补体激活进行特异性定量。通过 ELISA 评估补体激活，使用包被甘露聚糖来评估 LP，并使用包被 IgM 来评估 CP。 ELISA 显示，正常人血清 (NHS) 含有抗甘露聚糖的 IgG、IgA 和 IgM 抗体。这些抗体可能会诱导 CP 激活。使用 C1q 阻断和 MBL 阻断 mAb，证实 LP 和 CP 均有助于甘露聚糖激活补体。为了在不干扰 CP 的情况下量化 LP 活性，在存在 Clq 阻断 Ab 的情况下测量血清中的 LP 活性。通过 CP 激活包被 IgM 上的血清，导致 C1q、C4、C3 和 C5b-9 发生剂量依赖性沉积。这种激活和随后的补体沉积被 C1q 阻断 mAb 2204 和多克隆 Fab 抗 C1q Ab 完全抑制。在存在 mAb 2204 的情况下对 LP 进行的评估显示，使用来自 MBL 野生型 (AA) 而非 MBL 突变供体（AB 或 1313 基因型）的血清，C4、C3 和 C5b-9 出现强烈的剂量依赖性沉积，表明这些条件下的补体激活是 MBL 依赖性和 C1q 独立性的。使用新开发的寡核苷酸连接测定（OLA）来检测 MBL 外显子 1 多态性，从而鉴定出具有不同 MBL 基因型的供体。我们描述了一种新颖的功能测定，该测定能够通过全人血清中的 LP 定量自体补体激活，直至形成膜攻击复合物。该检测为患者诊断以及腰椎间盘突出疾病相关性的研究提供了新的可能性。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon I of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum.Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of Clq-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or 1313 genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms.We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP. (C) 2003 Elsevier Science Ltd. All rights reserved.