Arai Shiori, Orton E Christopher
Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado 80523-1678, USA.
J Heart Valve Dis. 2009 Jul;18(4):439-43.
The detergent-based 'decellularization' of xenogeneic tissues is one approach to scaffolding a tissue-engineered heart valve construct; however, concern persists regarding the immunogenicity of decellularized xenogeneic bioscaffolds. The study aims were to: (i) develop a sensitive and robust immunoblot-based assay for the detection of soluble protein antigens in xenogeneic bioscaffolds; and (ii) evaluate the completeness of protein antigen removal from sodium dodecyl sulfate (SDS)- or sodium deoxycholate (SD)-treated bovine pericardium (BP) or porcine aortic valve (PAV) conduit.
Homogenized BP or PAV were injected into rabbits to generate immune serum towards these tissues. Soluble proteins were extracted from untreated BP and PAV. Immunoblot analyses of the extracts were performed using pre-immune and 14-, 28-, 42-, 56- and 70-day post-immune serum. BP and PAV were treated sequentially with 4 h hypotonic lysis; with 0, 0.01, 0.025, 0.05, 0.1, 0.25 or 0.5% SDS or SD for 24 h; and with 96 h of aqueous wash-out. Immunoblot analyses of protein extracts from treated tissues were performed using 70-day post-immune rabbit serum.
Immunoblot analysis of untreated BP or PAV with pre-immune serum showed no immune banding. The immune banding density increased progressively when immunoblots were performed with 14-day through 70-day post-immune serum. The immunoblot analysis of treated BP and PAV showed that soluble protein antigen removal from SDS- or SD-treated tissues was incomplete.
Immunoblot analysis is a sensitive and robust assay for detecting soluble protein xenogeneic antigens after the decellularization of xenogeneic bioscaffolds. Under the study conditions, hypotonic lysis, SDS or SD detergent treatment, and aqueous wash-out-based decellularization of bovine pericardium and porcine aortic valve conduit did not completely remove detectable protein antigens.
基于去污剂的异种组织“去细胞化”是构建组织工程心脏瓣膜支架的一种方法；然而，对于去细胞化异种生物支架的免疫原性仍存在担忧。本研究的目的是：（i）开发一种灵敏且可靠的基于免疫印迹的检测方法，用于检测异种生物支架中的可溶性蛋白抗原；（ii）评估从经十二烷基硫酸钠（SDS）或脱氧胆酸钠（SD）处理的牛心包（BP）或猪主动脉瓣（PAV）导管中去除蛋白抗原的完整性。
将匀浆后的BP或PAV注射到兔子体内，以产生针对这些组织的免疫血清。从未经处理的BP和PAV中提取可溶性蛋白。使用免疫前血清以及免疫后14天、28天、42天、56天和70天的血清对提取物进行免疫印迹分析。BP和PAV依次进行4小时的低渗裂解；用0、0.01、0.025、0.05、0.1、0.25或0.5%的SDS或SD处理24小时；然后进行96小时的水洗脱。使用免疫后70天兔子血清对处理后组织的蛋白提取物进行免疫印迹分析。
用免疫前血清对未经处理的BP或PAV进行免疫印迹分析未显示免疫条带。当用免疫后14天至70天的血清进行免疫印迹分析时，免疫条带密度逐渐增加。对处理后的BP和PAV进行免疫印迹分析表明，从经SDS或SD处理的组织中去除可溶性蛋白抗原并不完全。
免疫印迹分析是一种灵敏且可靠的检测方法，用于检测异种生物支架去细胞化后的可溶性蛋白异种抗原。在本研究条件下，低渗裂解、SDS或SD去污剂处理以及基于水洗脱的牛心包和猪主动脉瓣导管去细胞化并未完全去除可检测到的蛋白抗原。
