Title: 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
Authors: Yueh, Sunny C. H.
Lai, Yi An
Chen, Wen Liang
Hsu, Hsiao Han
Mao, Simon J. T.
Institute of Biomedical Engineering
Keywords: human haptoglobin;affinity column;hemoglobin;apolipoprotien A-I;monoclonal antibody;sodium dodecyl sulfate
Issue Date: 15-Jan-2007
Abstract: 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
ISSN: 1570-0232
DOI: 10.1016/j.jchromb.2006.08.012
Volume: 845
Issue: 2
Begin Page: 210
End Page: 217
Appears in Collections:Conferences Paper

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