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.
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.
Yueh
Sunny C H
SC
Research Institute of Biochemical Engineering, College of Biological Science and Technology, National Chiao Tung University, Hsinchu 30050, Taiwan, ROC.
Lai
Yi An
YA
Chen
Wen Liang
WL
Hsu
Hsiao Han
HH
Mao
Simon J T
SJ
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
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D016632:Apolipoprotein A-I; C485359:HP protein, human; D006242:Haptoglobins; D006454:Hemoglobins