Complement Component C4d and Biomarkers of Transplantation Rejection


The complement system, a component of the innate immune system, consists of three pathways that participate in the recognition and clearance of pathogens. More recently, it has also been demonstrated to modulate adaptive immunity and bridge innate and adaptive immune responses. Dysregulation of this complex enzymatic system may cause inflammation as observed in autoimmune disease. Accurately assessing the activity of the complement system is an important diagnostic indicator of overall health.

In the classical and lectin pathways of complement activation, the C1r/C1s complex or MBL associated serine proteases cleave complement 4 (C4) to form C4a and C4b. As a result of C4 cleavage, an internal thiolester of C4b is exposed. This internal motif is highly reactive and will form a covalent bond with nearby protein or carbohydrate. This most often results in binding of C4b to an antibody or the surface of a cell such as vascular endothelial cells. Further cleavage of the C4b alpha chain by Factor I forms C4d and C4c, where C4d remains bound close to the site of C4b binding. The longer half-life of covalently bound C4d versus C4b makes it a perfect marker of complement activation long after weakly bound antibodies have been cleared by the blood stream (Sacks and Chowdhury 2002). For more information on all three complement pathways, read the complement system mini-review.

As a marker of complement activation, C4d has been used as an indicator in the evaluation of cellular and antibody mediated rejection in a variety of tissues such as kidney, heart, and pancreas. (Colvin and Smith 2005). Figure 1 shows C4d staining of human kidney in a patient with Lupus. The presence of C4d in renal peritubular capillaries has been found to be a key indicator for acute antibody mediated rejection (AMR) (Collins et al. 1999), shown in Figure 2 below, using the C4d polyclonal antibody. The C4d Rabbit Anti-Human Polyclonal Antibody (0300-0230) and Mouse Anti-Human C4d Antibody (2222-8004), clone 10-11, are ideal markers used in the detection of acute AMR for kidney, heart, pancreas, and lung allografts.

Rabbit anti Human C4d antibody  stained on human kidney from a patient with lupus erythematosus

Fig. 1. Formalin fixed paraffin embedded human kidney for a patient with Lupus erythematosus stained with Rabbit Anti-Human C4d Antibody (0300-0230) following sodium citrate (pH 6.0) mediated antigen retrieval.

Immunohistochemical C4d antibody staining of human kidney

Fig. 2. Immunohistochemical staining of human kidney with Rabbit Anti-Human C4d Antibody (0300-0230) showing localization to the peritubular capillaries.

C4d Antibodies

Description Clone  Applications Citations Code
Mouse Anti-Human C4d (Neoantigen)  057- IHC-F, E (1) MCA2649
Mouse Anti-Human C4d  10-11 IHC-F,  E,  IF, IHC-P*,  WB (9) 2222-8004
Mouse Anti-Human C4d  TDM16 E, IHC- P* (0) MCA5766GA
Rabbit Anti-Human C4d   IHC-F,  IF, IHC-P* (6) 0300-0230

Abbreviations: E, ELISA; IF, immunofluorescence; IHC-F, immunohistology - frozen sections; IHC-P, immunohistology - paraffin sections; WB, western blotting. 

Biomarkers of Transplantation Rejection

In addition to C4d, there are many other markers that can be used to monitor immunosuppression and ultimately act as an indicator of graft rejection. The table below lists a number of these markers for both acute and chronic allograft rejection in humans (Carpio et al. 2014 and Roedder et al. 2011), alongside cell type and organ expression. Many of these markers can be used on blood or urine thus avoiding the need for more invasive tissue sampling, using various techniques such as flow cytometry for example.

Allograft Rejection Markers.

Marker Cell Expression Organ Measurement as an Indication of Rejection Anti-Human Antibody
Acute Rejection        
C4d Endothelial cells Kidney   2222-8004 / 0300-0230 / MCA5766GA
CD138 B cells Kidney Antibody mediated MCA2459
FasLigand / CD178 T cells, NK cells Kidney Cytotoxic proteins MCA2409 / MCA2408GA
Granzyme B Cytolytic lymphocytes Kidney Cytotoxic proteins MCA2118 / MCA2119 / MCA2120
CXCL10      (IP-10) Neutrophils, epithelial cells, monocytes Kidney /lung / liver / heart Cytokine expression MCA5985GA / MCA1693
CXCL9 Dendritic cells, Microglia   Cytokine expression MCA6252
CD38 Monocytes, T cells, plasma cells Kidney Antibody mediated MCA1019
CD20 B cells Kidney T cell mediated MCA1710/ VPA00099
MICA T cells, NK cells Kidney Antibodies against non-HLA antigens MCA2403
PECAM-1 (CD31) Endothelial cells, platelets, granulocytes Kidney Proteogenomic  MCA1738
Chronic Rejection        
CCL2     (MCP-1) Monocytes, macrophages, Dendritic cells, Endothelial cells Kidney Antibody mediated MCA5981
CTGF Endothelial cells Kidney/ Heart Anti-fibrotic target AHP1278
TGF-beta Macrophages   Growth factor MCA797 / AHP1734

Table adapted from Roedder et al. 2011.


Other Related Complement Markers

In addition to C4d, Bio-Rad also offers a wide range of other anti-human markers for complement such as: