(2017), IXA 2017- Abstracts of the 14th Congress of the International Xenotransplantation Association, Baltimore, USA. Xenotransplantation, 24: n/a, e12328. doi:10.1111/xen.12328
The Optimization of Non-Gal Xeno-Antibodies Binding to Porcine Peripheral Blood Mononuclear Cells
Chengjiang Zhao1,2, Xuejun Ye1, Jiao Chen1, Chengjun Wang1, Hidetaka Hara3, David Cooper3, Yifan Dai4, Zhiming Cai1, Lisha Mou1.
1Shenzhen Xenotransplantation Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Afﬁliated Hospital of Shenzhen University, Shenzhen, People's Republic of China; 2Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, People's Republic of China; 3Xenotransplantation Program, Department of Surgery UAB, The University of Alabama at Birmingham, Birmingham , AL, United States; 4Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing , People's Republic of China
Shenzhen Xenotransplantation Research and Development Center.
Objective Non-Gal xeno antigens in recipients play an important role in the rejection of xenografts. We found that the traditional method was not sensitive enough to distinguish the variations of xeno antibodies in different human sera. The aim of this article is to optimize the detection of non-Gal xeno antigens to improve the sensitivity, accuracy and reliability. Methods The peripheral blood mononuclear cells (PBMCs) from Wuzhishan GGTA1 KO (GTKO) pig were incubated with pooled sera from 20 healthy people. Under the condition of different sera concentrations (5, 20, 100µL) and incubation time(0.5, 1, 2, 3, 6h), the IgM/IgG binding levels to GTKO PBMC was detected by flow cytometry. Results The IgM binding level to non-Gal PBMCs was significantly increased with the increase of incubation time, but the increase of IgG binding level had no statistic difference. It can also increase the IgM binding level to non-Gal PBMCs by increasing the incubation sera concentration, when the concentration of sera was 100µL and the incubation time was 3 hours, the IgM binding level was highest and had significant difference. Under the condition of same incubation time, only when the concentration of sera was up to 100µL and the incubation time was 6 hours, the IgG binding level had significant difference. In addition, extended the incubation time and increased the concentration of sera has no effect on the vitality of PBMCs. Conclusion Using 100µL human sera and incubation with non-Gal PBMCs for 3 hours was the best condition to detect the level of IgM antibody binding to non-Gal antigen. While, using 100µL human sera and incubation with non-Gal PBMCs for 6 hours was the best condition to detect the level of IgG antibody binding to non-Gal antigen. This optimization could be applied to select the donor pig with low level of no-Gal antigens.
This work has been supported by the following grants: Health and Family Planning Commission of Shenzhen Municipality (Grant number: 201501018), Sanming Project of Medicine in Shenzhen, Shenzhen Foundation of Science and Technology (Grant number: JCYJ20160229204849975), the Project of Shenzhen Engineering Center (GCZX2015043017281705), Fund for high level medical discipline construction of Shenzhen (Grant number: 2016031638)..
 Ezzelarab, M., H. Hara, J. Busch, P.P. Rood, X. Zhu, Z. Ibrahim, S. Ball, D. Ayares, M. Awwad, and D.K. Cooper, Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation, 2006. 13(5): p. 400-7.
 Azimzadeh, A.M., G.W. Byrne, M. Ezzelarab, E. Welty, G. Braileanu, X. Cheng, S.C. Robson, C.G. McGregor, D.K. Cooper, and R.N. Pierson, 3rd, Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells. Xenotransplantation, 2014. 21(6): p. 555-66.