Canaán-Haden L, Ayala M, Fernández-de-Cossio M E, Pedroso I, Rodés L, Gavilondo J V
Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Biotechniques. 1995 Oct;19(4):606-8, 610, 612 passim.
Immobilized metal affinity chromatography (IMAC) has been recently applied to the purification of of recombinant proteins bearing multi-histidine domains at their N or C terminus. We have now used this procedure for the single-step purification of an anti-Hepatitis B virus surface antigen (HBsAg) single-chain Fv (scFv) antibody fragment. Adjusting the metal ion (Cu+2 or Ni+2) and elution conditions (pH or imidazole), we efficiently separated active scFv forms from inactive molecules. Achieved purity was 93%, with a 20% yield with respect to the scFv content in the initial material. The pure scFv was coupled to CNBr-activated Sepharose 4B and compared the original monoclonal antibody (MAb) CB-Hep.1 in the immunoaffinity purification of a vaccine recombinant HBsAg (r-HBsAg). Results indicate that eluted antigen per mg of coupled ligand was similar for the scFv and the MAb when pure r-HBsAg was used as starting material. Preliminary results with unpurified starting material are also encouraging.
固定化金属亲和色谱法（IMAC）最近已应用于纯化在其N端或C端带有多组氨酸结构域的重组蛋白。我们现在已使用该方法对一种抗乙型肝炎病毒表面抗原（HBsAg）单链Fv（scFv）抗体片段进行单步纯化。通过调整金属离子（Cu²⁺或Ni²⁺）和洗脱条件（pH或咪唑），我们有效地从无活性分子中分离出了活性scFv形式。获得的纯度为93%，相对于初始材料中scFv的含量，产率为20%。将纯化的scFv偶联到CNBr活化的琼脂糖4B上，并在疫苗重组HBsAg（r-HBsAg）的免疫亲和纯化中与原始单克隆抗体（MAb）CB-Hep.1进行比较。结果表明，当使用纯r-HBsAg作为起始材料时，每毫克偶联配体洗脱的抗原对于scFv和MAb来说是相似的。使用未纯化起始材料的初步结果也令人鼓舞。
