Hylarides M D, Mallett R W, Meyer D L
NeoRx Corporation, 410 W. Harrison Street, Seattle, Washington 98119, USA.
Bioconjug Chem. 2001 May-Jun;12(3):421-7. doi: 10.1021/bc0001286.
The many uses of antibody-protein conjugates, especially antibody-streptavidin conjugates, give rise to the need for a reliable conjugation method offering reasonable yields and reproducible quality. We describe a method for preparing antibody-streptavidin conjugates that has consistently produced conjugates of quality and in sufficient quantity to be used in the clinical development and evaluation of the Pretarget delivery system. In this method antibody disulfides are reduced to generate reactive thiols, and maleimides are linked to streptavidin with the heterobifunctional cross-linking agent, SMCC. The two activated proteins are then mixed briefly before the conjugation is terminated with an oxidizing agent that reforms disulfides from unreacted thiols. The preponderance of the conjugate produced is 1:1 and 1:2 Ab:SA conjugate. This fraction is isolated from unconjugated proteins and high molecular weight byproduct by iminobiotin affinity and ion-exchange chromatography. The resulting conjugate is at least 90% 1:1 + 1:2 Ab:SA conjugate, contains no SA or Ab, and is produced reproducibly in 37% yield.
抗体 - 蛋白质偶联物，尤其是抗体 - 链霉亲和素偶联物的多种用途，使得需要一种可靠的偶联方法，该方法要有合理的产率和可重复的质量。我们描述了一种制备抗体 - 链霉亲和素偶联物的方法，该方法一直能产生高质量且数量充足的偶联物，可用于预靶向递送系统的临床开发和评估。在该方法中，抗体二硫键被还原以产生反应性硫醇，马来酰亚胺通过异双功能交联剂SMCC与链霉亲和素相连。然后将两种活化的蛋白质短暂混合，之后用一种氧化剂终止偶联反应，该氧化剂将未反应的硫醇重新形成二硫键。产生的偶联物中大部分是1:1和1:2的抗体:链霉亲和素偶联物。通过亚氨基生物素亲和和离子交换色谱从未偶联的蛋白质和高分子量副产物中分离出该部分。得到的偶联物至少90%是1:1 + 1:2的抗体:链霉亲和素偶联物，不含链霉亲和素或抗体，并且以37%的产率可重复生产。
