Paek S H, Bachas L G, Schramm W
Reproductive Sciences Program, University of Michigan, Ann Arbor 48109.
Anal Biochem. 1993 Apr;210(1):145-54. doi: 10.1006/abio.1993.1165.
We investigated the synthesis of progesterone-horseradish peroxidase (P-HRP) conjugates and products purified by affinity chromatography. The obtained preparations were characterized with an immobilized monoclonal antibody in solid-phase immunoassays. Three homogeneous P-HRP conjugates were isolated. Two preparations were identified to contain a single progesterone ligand on the enzyme molecule. A third preparation contained two progesterone ligands. We postulate that conjugation can occur at two different positions on the enzyme, and that the different microenvironment of the protein structure surrounding the ligand contributes to different binding constants of the conjugates with immunoglobulin. By comparing the effective binding constants derived from affinity chromatography and from Scatchard analysis, we have demonstrated that the divalent conjugate binds to antibody immobilized on planar surfaces only by a single attachment due to steric restriction. Dose-response curves for progesterone using the isolated P-HRP conjugates have been investigated and compared.
我们研究了孕酮 - 辣根过氧化物酶（P - HRP）偶联物的合成以及通过亲和色谱法纯化的产物。在固相免疫测定中，用固定化单克隆抗体对所获得的制剂进行表征。分离出三种均一的P - HRP偶联物。鉴定出两种制剂在酶分子上含有单个孕酮配体。第三种制剂含有两个孕酮配体。我们推测偶联可能发生在酶上的两个不同位置，并且配体周围蛋白质结构的不同微环境导致偶联物与免疫球蛋白的结合常数不同。通过比较源自亲和色谱法和Scatchard分析的有效结合常数，我们证明由于空间位阻，二价偶联物仅通过单一附着与固定在平面表面上的抗体结合。已经研究并比较了使用分离的P - HRP偶联物的孕酮剂量 - 反应曲线。
