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：Sunny C.H. Yueh ; Yi An Lai ; Wen Liang Chen ; Hsiao Han Hsu ; Simon J.T. Mao
- 关键词：Human haptoglobin ; Affinity column ; Hemoglobin ; Apolipoprotien A-I ; Monoclonal antibody ; Sodium dodecyl sulfate
- 刊名：Journal of Chromatography B ; Analytical Technologies in the Biomedical and Life Sciences
- 出版年：2007
- 期刊代码：124_15700232
- 类别：ch
- 出版时间：15 January 2007
- 卷：845
- 期：2
- 页码：210-217
- 文件大小：651 K
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 β-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 α-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.