Anti‐pig antibody levels in naïve baboons and cynomolgus monkeys

Abstract

Anti-pig antibodies (APA) were analysed in serum from 28 naïve wild-caught baboons (originating from Kenya) and 31 naïve captive-bred cynomolgus monkeys (13 from the Philippines and 18 from Mauritius), using a haemolytic assay with pig erythrocytes (APA), flow cytometry on the porcine lymphoma T-cell cell line L35, and enzyme linked immunosorbent assay (ELISA) using alpha-Gal type II and type VI antigen. This was extended in baboon samples by the evaluation in two laboratories (Imutran, Cambridge, UK and Immerge, Boston, USA), and by antibody absorption using either immobilized alpha-Gal type II or alpha-Gal type VI. Anti-porcine antibodies were demonstrated in all assays with substantial variability within and between the three non-human primate groups. Immunoglobulin (Ig)M antibody levels tended to be similar to or higher than those in a pooled normal human standard serum while IgG levels tended to be lower. Highest antibody levels were recorded in Mauritius cynomolgus monkeys. There were statistically significant correlations between assays for IgM or IgG class anti-Gal antibodies using either alpha-Gal type II or alpha-Gal type VI as antigen, both for different assays and two laboratories involved. Also, significant correlations were observed between the anti-Gal and L35 binding assays. Baboon sera before and after absorption to immobilized alpha-Gal type II or type VI were analysed for anti-Gal type VI or type II antibody: levels were almost undetectable indicating that most anti-Gal antibodies react to epitopes shared between alpha-Gal type II and type VI oligosaccharides. Finally, the relation between APA and outcome of porcine heart xenotransplantation in cynomolgus monkeys and baboons showed no apparent relation between pre-transplant APA levels and the occurrence of hyperacute rejection (HAR) when compared with non-immunological cause of organ/recipient dysfunction or acute humoral xenograft rejection during the first 4 days post-transplantation or survival exceeding 4 days post-transplantation.

Elicited Antibodies in Baboons Exposed to Tissues from ??1,3-Galactosyltransferase Gene-Knockout Pigs

“…Complete removal of anti-Gal Ab (to below the detection limit of the assay, i.e., 5 g/ml for IgM and 0.5 g/ml for IgG) was confirmed by an anti-Gal enzyme-linked immunosorbent assay (14,15).…”

Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity

“…SureBlue substrate (Kirkegaard & Perry Laboratories) was then applied, the reaction was stopped by the addition of 0.2 M H 2 SO 4 , and absorbance was measured on a Packard SpectraCount ELISA plate reader with PlateReader version 3.0 software (Packard Biosciences). For each duplicate sample, a mean absorbance vs dilution curve was plotted, and the area under the curve was calculated (31). The area of the curve of an experimental sample was expressed as the percentage of positive control (pooled hyperimmune serum).…”

Potential of an Injectable Polymer to Prevent Hyperacute Rejection of Ex Vivo Perfused Porcine Lungs

“…The two most common structural forms of ␣-Gal occurring are the trisaccharides type II and type VI. In naive baboons almost all antibodies are directed against these two epitopes, and extensive cross-reactivity has been reported (29). Primates have a polymorphism of the anti-␣-Gal antibody population against ␣-Gal disaccharides, trisaccharides one, two, and six and pentasaccharides (30).…”