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Published on: April 23, 2010
Immunoblot detection of soluble protein antigens from sodium dodecyl sulfate- and sodium deoxycholate-treated
Shiori Arai1, E Christopher Orton
1Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado 80523-1678, USA.
Background And Aims Of The Study:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
