Detection of xenoantibodies using a simple flow cytometric assay

Higher primates, including humans, have high levels of preexisting naturally circulating antibodies that predominantly recognize the epitope Gal (1,3-Gal), which is highly expressed on the surface of xenogenic cells. Deposition of these antibodies on the endothelial cell surface of vascularized xenografts leads to an activation of the classical pathway of the complement system, resulting in tissue ischemia and necrosis with rapid demise of the xenograft. This hyperacute rejection (HAR) is always a major barrier in xenograft transplantation and should be minimized by accurately monitoring the naturally occurring antibodies. In the present study, we utilized a simple and rapid flow cytometric (FCM) assay to monitor the presence of these naturally occurring antibodies. We found that the FCM assay is very effective in measuring human antibodies bound to the xenogenic cells, which cause cytotoxicity. This assay could be useful in the pre- and post-xenotransplantation monitoring of xenoantibodies, thus, helping in the development of strategies to block the binding of preformed human antibodies to the xenograft in order to overcome the problem of HAR.