Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies

Abstract

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.

Keywords: C4d staining; antibody-mediated rejection; complement C4d; immunohistochemistry.

Figure 1

C4d visualization in rejected kidney graft tissues. Left panel: The C4d immunostaining of chosen tissue sections (rows A–F) with rabbit polyclonal anti-C4d antibody (9aa), monoclonal XS43, and SP91. The values presented in the pictures are the scores for a specific reading vs. background, e.g., 2/0. Middle panel: The slides presented in in row (D) of the left panel are shown at a higher magnification (400×) to better illustrate differences between the C4d staining considered specific (a lineage of blood vessels, blue arrowheads) and nonspecific (cytoplasmic staining in epithelial cells of renal tubules, red arrowheads). Right panel: The scoring of the specific C4d signal (left column) and background signal (right column) in 106 tissue microarrays from kidney graft rejections. The readout obtained for the staining with the SP91 clone as used for the routine IVD procedure was considered as a reference. The color code represents an improved (green, in the case of enhancement in a specific signal or lowering the background) or worsened (red, in the case of a background increase) score vs. the SP91 reference.