Scheiner O, Stemberger H, Kraft D, Wiedermann G
J Immunol Methods. 1978;21(3-4):355-64. doi: 10.1016/0022-1759(78)90162-x.
Anti-BPO antibodies were purified by means of affinity chromatography using AH-Sepharose 4B coated with covalently bound BPO groups. Specific elution was achieved by the hapten analogue BPO-epsilon-aminocaproic acid (BPO-EACA); desorption of the remaining antibody was performed thereafter by 0.1 M acetic acid. The resulting antibody fractions--hapten-eluted antibody (H-Ab) and acid eluted antibody (A-Ab), respectively--were further separated by ion-exchange chromatography which led to the appearance of 3 subfractions in the case of H-Ab (H1, H2, H3) and 2 subfractions in the case of A-Ab (A1 and A2). In liquid isoelectrofocusing an inhomogeneous pattern resulted. The bulk of antibodies focused between pH 6.5 and 7.0. The average avidity of H-Ab was found to be higher than that of A-Ab suggesting that avidity may influence the elution pattern in affinity chromatography. The hydrophobic influence of the "spacer" and/or interactions of antibodies directed against the hydrophobic regions of the BPO group may explain why a considerable part of the antibodies could be recovered from the immunosorbent only by acid elution.
抗BPO抗体通过亲和层析法进行纯化，使用共价结合有BPO基团的AH-琼脂糖4B。通过半抗原类似物BPO-ε-氨基己酸（BPO-EACA）实现特异性洗脱；随后用0.1M乙酸进行剩余抗体的解吸。所得抗体组分——分别为半抗原洗脱抗体（H-Ab）和酸洗脱抗体（A-Ab）——通过离子交换层析进一步分离，结果H-Ab出现3个亚组分（H1、H2、H3），A-Ab出现2个亚组分（A1和A2）。在液相等电聚焦中得到了不均匀的图谱。大部分抗体聚焦在pH 6.5至7.0之间。发现H-Ab的平均亲和力高于A-Ab，这表明亲和力可能会影响亲和层析中的洗脱模式。“间隔臂”的疏水作用和/或针对BPO基团疏水区域的抗体相互作用，可能解释了为什么相当一部分抗体只能通过酸洗脱从免疫吸附剂中回收。
