Characterization and application of two novel monoclonal antibodies against CD40L: epitope and functional studies on cell membrane CD40L and studies on the origin of soluble serum CD40L

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Abstract
CD40 ligand, a 33‐kDa cell membrane molecule, a member of the tumor necrosis factor superfamily, is an important costimulatory molecule during immune response. Here, we report on two functional mouse anti‐human CD40L monoclonal antibodies 1B1 and 4F1 characterized by flow cytometry, Western blotting, and competition assay. The antibodies bound to distinct CD40L epitopes and therefore resulted in different bioactivity. Both antibodies could induce CD4+ T‐cell alloantigenic hyporesponsiveness ex vivo. The antibodies were matched to develop a two‐site enzyme‐linked immunosorbent assay (ELISA) for soluble CD40L (sCD40L). Using this ELISA assay, we found major differences between plasma and serum sCD40L levels. Because the count of platelet sharply decreased in aplastic anemia (AA) and idiopathic thrombocytopenic purpura (ITP), we further analyzed the sCD40L concentration in the plasma of AA and ITP patients. The results showed that the sCD40L in serum was much lower than that of healthy subjects. These data demonstrate that platelets seem to be a major contributor to sCD40L, though not the only source of sCD40L in serum.