Pharmaceutical Biotechnology 2015 22133 ~ 37 33 ELISA α-gal * 250101 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 ± 0. 73 10 5 ~ 4. 7 ± 0. 85 10 5 P < 0. 05 α-gal P < 0. 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 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 4 5-8 1. 6 10-6 mol /L - 20 9 SP2 /0 α-gal M86 10% RPMI-1640 GIBCO-BRL 10 α-gal 11 8 12 IgM 13 α-gal 1 PBS ph 7. 41% BSA 0. 01 g BSA 37 5% CO 2 HRP α-gal 0. 05% Tween-20 TMB α-gal α-gal α-gal Bertin Bio-Rad * 2014-04-30 2014-12-09 No. 2012BAI22B01 PI No. 2012NELRMD007 No. 2014GSF118151 No. ZR2014CQ041 1982- E-mailsunzx@ 163. com
34 22 1 IKA Sigma 500 100 μl / 37 1 h Eppendorf 0. 000 01 g 2 2. 1 3 A M86 % = A A /A B 100% 5 500 mg 8 3 mm 1 5 mm 5 000 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% 3 2. 5. 3 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 6 800 r /min 30 s / 2 2. 6 1% BSA 1 ml 2. 1 ± Student's t P < 0. 05 2. 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. 4 ELISA 1% BSA 1 125 α-gal 200 μl / 4 / M86 SP2 /0 α-gal α-gal M86 1% BSA M86 50 100 μl / SP2 /0 4 M86 M86 α-gal 1 M86 30 s 3 SP2 /0 α-gal M86 14 000 r /min 5 min 100 μl / α-gal-m86 M86 100 μl / 37 2 h α- Gal-M86-30 s 3 1% BSA HRP TMB 100 μl / 37 30 min 100 μl / 37 15 min A 492 2. 5 3 SP2 /0 α-gal x ELISA M86 y R 2 = 0. 991 5 1 1 SP2 /0 2 α-gal M86
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 /0 10 6 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 4 3. 3 ELISA α-gal 4 ELISA α-gal α-gal M86 α-gal α- gal M86 α-gal M86 ELISA α-gal α-gal ELISA
36 22 1 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 4 5-8 α-gal Gal SP2 /0 SP2 /0 Galili U 10 α-gal M86 ELISA α-gal 6 ELISA 10 SP2 /0 10 6 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 2012 93 7686-692. α-gal 2Galili URachmilewitz EAPeleg A et al. A unique natural human IgG antibody with anti-alpha-galactosyl specificityj. J Exp Med 1984 1605 1519-1531. α-gal 3Choi HJKim MKLee HJ et al. Effect of alphagal on corneal α-gal xenotransplantation in a mouse modelj. Xenotransplantation α-gal 2011 183 176-182. 4 4Himaki 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 2010 566 630-638. 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 1994 263 1398. 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 1993 566 1433-1442. ELISA 7Naso F Gandaglia A Iop L et al. Alpha-Gal detectors in xenotransplantation researcha word of cautionj. Xenotransplanta-
ELISA α-gal 37 tion 2012 194215-220. 8Shao Y Yu Y Pei CG et al. The expression and distribution of alpha-gal gene in various species ocular surface tissuej. Int J Ophthalmol 2012 55 543-548. 9McKenzie IF Xing PX Vaughan HA et al. Distribution of the major xenoantigen gal alpha 1-3galfor pig to human xenograftsj. Transpl Immunol 1994 22 81-86. 10Galili U LaTemple DC Radic MZ. 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. Elimination of alpha-gal xenoreactive epitopealpha-galactosidase treatment of porcine heart valvesj. J Heart Valve Dis 2012 213 387-397. 12Feng W Fu L Liu J et al. The expression and distribution of xenogeneic targeted antigens on porcine bone tissuej. Transplant Proc 2012 445 1419-1422. 13Naso 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 2011 74 1728-1734. 14Galili 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 1988 263 3317755-17762. 15Macher BAGalili U. The Galalpha1 3Galbeta1 4GlcNAc-R alpha-gal epitopea carbohydrate of unique evolution and clinical relevancej. Biochim Biophys Acta 2008 17802 75-88. 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 250101 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 ±0. 73 10 5 /mg ~ 4. 7 ± 0. 85 10 5 /mg which is obviously less than the same weight of raw bone tissue. Differences between groups were statistically significant P < 0. 05. The expression of α-gal on Skin repair membrane is also less than its raw leather tissue P < 0. 05. 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 2015-6 5 40 2015-7 HCV HCV