Pharmaceutical Biotechnology ~ 37. α-gal α-gal ELISA A α-gal α-gal M86 Alexis 37 5% CO 2

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1 Pharmaceutical Biotechnology ~ ELISA α-gal * α-gal Galactosyl α-1 3-galactosyl β-1 4-N-acetylglucosaminyl α-gal epitopes α-gal α-gal M86 ELISA α-gal α-gal 3. 9 ± ~ 4. 7 ± P < α-gal P < ELISA ELISA α-gal α-gal α-gal α-gal ELISA Q503 A ELISA ELISA Inhibition assay Galili U 2 α-gal α-gal IgG IgG 1% 1 α-gal Galactosyl α-1 3-galactosyl β-1 4-Naectylglucosaminyl α-gal epitopes 1 3 Choi HJ 3 α-gal 10 ml 1 PBS 4 α-gal α-gal M86Alexis - 20 α-gal-bsa Dextra Reading UK α-gal Dextra 0. 5 mg gal 2mL mol /L SP2 /0 α-gal M86 10% RPMI-1640 GIBCO-BRL 10 α-gal IgM 13 α-gal 1 PBS ph 7. 41% BSA g BSA 37 5% CO 2 HRP α-gal 0. 05% Tween-20 TMB α-gal α-gal α-gal Bertin Bio-Rad * No. 2012BAI22B01 PI No. 2012NELRMD007 No. 2014GSF No. ZR2014CQ sunzx@ 163. com

05 ELISA ELISA α-gal α-gal α-gal α-gal ELISA Q503 A 1005-8915201501-033-05 ELISA ELISA Inhibition assay 1 1984 Galili U 2 α-gal α-gal IgG IgG 1% 1 α-gal Galactosyl α-1 3-galactosyl β-1

2 IKA Sigma μl / 37 1 h Eppendorf g A M86 % = A A /A B 100% mg 8 3 mm 1 5 mm r /min M86 A A A 492 A B M86 30 s / 2 A 492 1% BSA 1 ml x 28 M86 y 20 μl 1. 5 ml 80 μl /1% BSA R 20% M86 IC 50 50% % 2. 2 α-gal /mg= 1 cm 2 A /B / 1. 5 ml α-gal A50% 24 h B50% 8 3 mm 1 5 mm r /min 30 s / % BSA 1 ml 2. 1 ± Student's t P < P < ** P < μl 1% BSA SP2 /0 20 μl 8 80 μl / 3. 1 ELISA 1% BSA 20% Galili U 14 SP2 / α-gal SP2 / ELISA 1% BSA α-gal 200 μl / 4 / M86 SP2 /0 α-gal α-gal M86 1% BSA M μl / SP2 /0 4 M86 M86 α-gal 1 M86 30 s 3 SP2 /0 α-gal M r /min 5 min 100 μl / α-gal-m86 M μl / 37 2 h α- Gal-M86-30 s 3 1% BSA HRP TMB 100 μl / min 100 μl / min A SP2 /0 α-gal x ELISA M86 y R 2 = SP2 /0 2 α-gal M86

3 P < 0. 05 ** P < 0. 01 40 μl 1% BSA 1 10 6 SP2 /0 20 μl 8 80 μl / 3. 1 ELISA 1% BSA 20% Galili U 14 SP2 /0 10 6 α-gal SP2 /0 3 2.

3 ELISA α-gal 35 α-gal 5 ELISA α-gal α-gal α-gal ELISA ELISA α-gal Fig 1 The standard curve of ELISA inhibition assay with number of α-gal epitope expression on SP2 /0 cells Fig 3 α-gal epitope expression on osseous tissue Fig 2 The standard curve of ELISA inhibition assay with tissue volume concentration Fig 4 α-gal epitope expression on skin 3. 2 α-gal ELISA α-gal α-gal M86 α-gal 50% 10 SP2 / α-gal α-gal 3 Fig 5 The recovery of α-gal epitope by α-gal ELISA inhibition assay α-gal ELISA α-gal α-gal ELISA α-gal 4 ELISA α-gal α-gal M86 α-gal α- gal M86 α-gal M86 ELISA α-gal α-gal ELISA

concentration Fig 4 α-gal epitope expression on skin 3.

4 α-gal SP2 /0 α-gal 1% B ELISA α-gal α-gal α-gal α-gal IgG IgG 1% 2 α-gal 90% 15 α-gal α-gal α-gal α-gal Gal SP2 /0 SP2 /0 Galili U 10 α-gal M86 ELISA α-gal 6 ELISA 10 SP2 / α- α-gal α- Gal M86 α-gal HRP α-gal ELISA M86 ELISA AELISA inhibition assaybtraditional ELISA assay Fig 6 The flow chart of ELISA α-gal 1McGregor CG Ricci D Miyagi N et al. Human CD55 expression blocks hyperacute rejection and restricts complement activation in 3 Gal knockout cardiac xenograftsj. Transplantation α-gal 2Galili URachmilewitz EAPeleg A et al. A unique natural human IgG antibody with anti-alpha-galactosyl specificityj. J Exp Med α-gal 3Choi HJKim MKLee HJ et al. Effect of alphagal on corneal α-gal xenotransplantation in a mouse modelj. Xenotransplantation α-gal Himaki TWatanabe S Chi H et al. Production of genetically M86 modified porcine blastocysts by somatic cell nuclear transfer preliminary results toward production of xenograft-competent mini- pigsj. J Reprod Dev M86 ature α-gal 5Oriol R Ye Y Koren E et al. Carbohydrate antigens of vascular endothelium and other pig tissues reacting with human natural antibodiesj. Transplant Proc α-gal ELISA 6Oriol R Ye Y Koren E et al. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for α-gal hyperacute vascular rejection in pig-to-man organ xenotransplantationj. Transplantation ELISA 7Naso F Gandaglia A Iop L et al. Alpha-Gal detectors in xenotransplantation researcha word of cautionj. Xenotransplanta-

Human CD55 expression blocks hyperacute rejection and restricts complement activation in 3 Gal knockout cardiac xenograftsj. Transplantation 2012 93 7686-692.

5 ELISA α-gal 37 tion Shao Y Yu Y Pei CG et al. The expression and distribution of alpha-gal gene in various species ocular surface tissuej. Int J Ophthalmol McKenzie IF Xing PX Vaughan HA et al. Distribution of the major xenoantigen gal alpha 1-3galfor pig to human xenograftsj. Transpl Immunol Galili U LaTemple DC Radic MZ. A sensitive assay for measuring alpha-gal epitope expression on cells by a monoclonal anti- Gal antibodyj. Transplantation Choi SY Jeong HJ Lim HG et al. Elimination of alpha-gal xenoreactive epitopealpha-galactosidase treatment of porcine heart valvesj. J Heart Valve Dis Feng W Fu L Liu J et al. The expression and distribution of xenogeneic targeted antigens on porcine bone tissuej. Transplant Proc Naso FGandaglia A Iop L et al. First quantitative assay of alpha-gal in soft tissuespresence and distribution of the epitope before and after cell removal from xenogeneic heart valvesj. Acta Biomater Galili U Shohet SB Kobrin E et al. Man apes and Old World monkeys differ from other mammals in the expression of alphagalactosyl epitopes on nucleated cellsj. J Biol Chem Macher BAGalili U. The Galalpha1 3Galbeta1 4GlcNAc-R alpha-gal epitopea carbohydrate of unique evolution and clinical relevancej. Biochim Biophys Acta Determination of α-gal Epitope in Animal-Derived Biomaterials Using ELISA Inhibition Assay SUN Xiao-xia LIU Jia LIU Cheng-hu HOU Li Shandong Quality Inspection Center for Medical DevicesShandong Provincial Key laboratory of Biological Evaluation of Medical Devices Jinan China Abstract Animal-derived biomaterials with abundant sources and excellent functions are widely used in clinical treatment but high expression of α-gal antigens Galactosyl α-1 3-galactosyl β-1 4-N-acetylglucosaminyl α-gal epitopeson these biomaterials could bind to the human specific α-gal antibody resulting in graft failure with hyperacute graft rejection and chronic immune toxicity. In this study the common clinical animal-derived biomaterials as objects were used to quantify their α-gal epitope by ELISA inhibition assay with a monoclonal anti-gal antibody. The results show that the expression of α-gal on two biological bone is 3. 9 ± /mg ~ 4. 7 ± /mg which is obviously less than the same weight of raw bone tissue. Differences between groups were statistically significant P < The expression of α-gal on Skin repair membrane is also less than its raw leather tissue P < Meanwhile the ELISA inhibition assay has good recovery efficiency. It is suggested that ELISA inhibition assay is a quick and specific method for α-gal epitope detection on xenograft material which provides experimental basis for α-gal-related xenotransplantation research and clinical application. Key words α-gal epitope α-gal antibody ELISA inhibition assayanimal-derived biomaterialsxenograft rejection Chronic immune toxicity 櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗 HCV HCV

A sensitive assay for measuring alpha-gal epitope expression on cells by a monoclonal anti- Gal antibodyj. Transplantation 1998 658 1129-1132. 11Choi SY Jeong HJ Lim HG et al.

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