The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells

Abstract

Background The results of the assay for measuring anti-nonGal antibodies (which affect pig xenograft survival) in recipients is important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-nonGal antibody binding to porcine aortic endothelial cells (pAECs). Materials and Methods Pooled human, naive and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, 3h) and concentrations (5, 10, 20, 40μL) was determined by flow cytometry. Results An increase in incubation time from 30min to 2h was associated with increases in anti-nonGal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<0.05). Pooled human serum showed a significant increase in anti-nonGal IgM (x1.5), and a minimal increase in anti-nonGal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2h incubation was ×1.5 and ×2 greater than after 30min incubation, respectively whereas naïve baboon sera showed minimal (non-significant) increase in anti-nonGal IgM/IgG antibody binding. With 2h incubation, increasing the serum concentration from 5μL to 20μL significantly increased antibody binding to nonGal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Conclusions Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-nonGal antibodies, without affecting cell viability in vitro.

Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation

“…). These findings were expected from previous studies 28,33,34 . WT and GTKO pAECs were positive for CD31 (vascular endothelial cell) expression (> 90%) ( Supplementary Fig.…”

“…WT and GTKO pAECs were positive for CD31 (vascular endothelial cell) expression (> 90%) ( Supplementary Fig. 4B), as previously described 28 .…”

“…Briefly, blood was washed × 3 with phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA), and centrifuged for 5 min at 4 °C at 910 g. The washed RBCs were suspended in (i) fluorescence-activated cell sorting (FACS) buffer [PBS containing 1% bovine serum albumin (BSA) and 0.1% NaNH 3 ] for surface staining, or (ii) diluted to 10 × 10 6 /ml (1 × 10 6 /100 µl) in PBS at room temperature for IgM and IgG binding, or (iii) diluted to 800 × 10 6 /ml in PBS at room temperature for complement-dependent hemolytic assay. pAECs were isolated from fresh pig aortas and cultured, as previously described [26][27][28] .…”

“…pAECs were diluted to 1 × 10 5 cells per tube in FACS buffer. Surface expression of Gal, Neu5Gc, and Sd a antigens was detected by flow cytometry, as previously described 26,28,29 .…”

B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy

“…It included (i) flow cytometry to monitor T (CD3 + T) and B (CD22 + B) cell numbers in the blood (to determine the effect of ATG and anti-CD20mAb) (CD22 was used as a B cell marker because all baboons were administered anti-CD20mAb); (ii) flow cytometry to monitor binding of baboon xenoreactive anti-nonGal antibodies (IgM and IgG) to GTKO/CD46 pig aortic endothelial cells (pAECs), pig red blood cells (RBCs), and pig peripheral blood mononuclear cells (PBMCs). Binding of IgM and IgG was assessed using relative geometric mean (GM), which was calculated by dividing the geometric mean for each sample by the negative control [29–31]. ; (iii) carboxyfluorescein diacetate succinimidyl esterase (CFSE)-mixed lymphocyte reaction (MLR) pre-transplantation and at euthanasia using GTKO/CD46 pig PBMCs as stimulators and baboon PBMCs as responders, as previously described [8,22,32].…”