Department of Cell Biology and Immunology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Dębinki 1 Street, 80-211 Gdańsk, Poland.
Department of Pathomorphology, Medical University of Gdańsk, Smoluchowskiego 17 Street, 80-214 Gdańsk, Poland.
Int J Mol Sci. 2024 Sep 30;25(19):10526. doi: 10.3390/ijms251910526.
C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.
C4d 是激活补体成分 C4b 的末端降解产物，在经典和凝集素补体途径的早期步骤中出现。在 C4d 的一级序列中，存在一个反应性硫酯基团，它与附近的表面共价结合，从而标记补体激活的位置。这一特性使 C4d 成为免疫组织化学染色的目标，旨在帮助诊断，除其他外，移植器官的抗体介导排斥反应、膜性肾小球肾炎、大疱性类天疱疮或炎性肌病。然而，C4d 免疫染色的可信度存在争议，因为一些现有的抗 C4d 抗体在周围组织和体液中存在高背景，并且在目标结构中沉积模式扩散。在此，我们展示了一种改良的兔抗 C4d 抗体，最初是针对该补体片段的 C 末端线性新表位产生的。通过 ELISA 测量，与 C4b 和天然 C4 蛋白的交叉反应性较小，以及在福尔马林固定石蜡包埋材料的免疫组织化学分析中获得特异性信号所需的相对较低浓度，使改良抗体优于市售的用于体外诊断的兔单克隆抗 C4d 抗体 SP91，这通过对一系列肾移植活检的染色得到证明。
