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.
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 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.
authors.affiliation: Research Institute of Biochemical Engineering, College of Biological Science and Technology, National Chiao Tung University, Hsinchu 30050, Taiwan, ROC.
authors.forename: Sunny C H
authors.initials: SC
authors.lastname: Yueh
authors.forename: Yi An
authors.initials: YA
authors.lastname: Lai
authors.forename: Wen Liang
authors.initials: WL
authors.lastname: Chen
authors.forename: Hsiao Han
authors.initials: HH
authors.lastname: Hsu
authors.forename: Simon J T
authors.initials: SJ
authors.lastname: Mao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex; D016632:Apolipoprotein A-I / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D006242:Haptoglobins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D016632:Apolipoprotein A-I; C485359:HP protein, human; D006242:Haptoglobins; D006454:Hemoglobins