Identification of porcine membrane antigens involved in the cytotoxic response mediated by human xenoreactive antibodies

Abstract

The identification of xenoantigens on the surface of endothelial cells is important for understanding the mechanism of hyperacute rejection and development of abrogating methods. The objective of our study was to identify the porcine antigens that, when bound by xenoreactive antibodies in human serum, result in cytotoxicity of porcine cells. Human AB and O sera were adsorbed with porcine aortae, erythrocytes, platelets, and a broad spectrum of immobilized carbohydrate moieties (Synsorbs). Aortae and erythrocytes were able to adsorb the xenoreactive antibodies that were cytotoxic to porcine cells (LLC‐PK1), determined using an MTT cytotoxicity assay. Only carbohydrates having the αGal(1–3)βGal(1–4) moieties (Synsorbs 90, 115) were able to significantly reduce cytotoxicity with both types of sera. Western blots of porcine aortic endothelial cells (PAEC) and LLC‐PK1 cell membrane extracts probed with unadsorbed sera indicate the binding of xenoreactive IgM to approximately 17 and 11 antigen bands, respectively, having molecular weights ranging from 20–133 kDa. Anti‐IgG development showed 8 and 11 antigen bands on PAEC and LLC‐PK1 membrane preparations, respectively. When blots were performed using adsorbed AB sera, the binding to all antigens was still observed. When Synsorb 90 bound antibodies were used to probe the blots, the majority of antigens were still detected. This suggests that the binding of xenoantibodies to the most prominent antigens, as detected by Western blot procedures may not be the ones to which cytotoxic xenoreactive antibodies bind. Alternative approaches are required to identify such antigens.

Multivalent Galα1,3Gal‐substitution makes recombinant mucin–immunoglobulins efficient absorbers of anti‐pig antibodies

“…The higher resolving power of two‐dimensional IEF/SDS‐PAGE in combination with Western blot analysis (Fig. 6), allowed us to show that the complexity of PAEC antigens reacting with human natural antibodies is greater than previously shown by one‐dimensional SDS‐PAGE and Western blotting [57,59]. It is clear that IgM and IgG in pooled human blood group AB serum have overlapping, yet distinct, specificities.…”

“…In both platelets and ECs, the integrin family of adhesion molecules were shown to carry Galα1,3Gal epitopes, and to be major binders of human xenoreactive antibodies that was removed following perfusion through a pig kidney [55,57]. Others have also investigated the binding of human natural IgG and IgM antibodies to porcine endothelial cell membrane components, and in Western blot experiments approximately 17 PAEC membrane components reacted with human IgM and eight components with human IgG [59]. The higher resolving power of two‐dimensional IEF/SDS‐PAGE in combination with Western blot analysis (Fig.…”

Humoral Human Xenoreactivity Against Isolated Pig Pancreatic Islets

“…23 Therefore, a number of investigators recommend that AB sera be used for studies investigating human xenoreactive antibodies to porcine cells. 19,24 Thus, we used AB sera in this study to exclude the effect of blood group antibodies. Furthermore, we did not add the serum of any other species, such as fetal calf serum or fetal bovine serum, to the culture medium during the study to avoid any influence of xenogeneic antibodies in the fetal serum.…”

Removal of xenoreactive antibodies by protein‐A immunoadsorption: experience in 22 patients

“…All assays were performed in duplicate with a negative control consisting of cells treated with complement in the absence of human serum and a positive control consisting of cells treated with 70% ethanol. Data were expressed as percentage of cytotoxicity, which was calculated as follows: % cytotoxicity =[1‐(test OD/control OD)] × 100 [40].…”