An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate.

ASIA unversity > 醫學暨健康學院 > 生物科技學系 > 期刊論文 > Item 310904400/4439

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题名:	An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate.
作者:	Yueh CH;Lai YA;Hsu HH;Chen WL;Mao SJT
贡献者:	Department of Biotechnology
日期:	2007-01
上传时间:	2009-11-26 01:43:10 (UTC+0)
出版者:	Asia University
摘要:	Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp beta-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against alpha-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. As a result, the isolated Hp was devoid of apoA-I and was able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail.
關聯:	Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 845(2):210-7
显示于类别:	[生物科技學系] 期刊論文

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