標題: 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, Sunny C. H.
Lai, Yi An
Chen, Wen Liang
Hsu, Hsiao Han
Mao, Simon J. T.
生醫工程研究所
Institute of Biomedical Engineering
關鍵字: human haptoglobin;affinity column;hemoglobin;apolipoprotien A-I;monoclonal antibody;sodium dodecyl sulfate
公開日期: 15-一月-2007
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 a-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-HC1, pH 6.8. As a result, the isolated Hp was devoid of apoA-1 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. (c) 2006 Elsevier B.V. All rights reserved.
URI: http://dx.doi.org/10.1016/j.jchromb.2006.08.012
http://hdl.handle.net/11536/5334
ISSN: 1570-0232
DOI: 10.1016/j.jchromb.2006.08.012
期刊: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume: 845
Issue: 2
起始頁: 210
結束頁: 217
顯示於類別:會議論文


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