Terpetschnig E, Szmacinski H, Lakowicz J R
Department of Biological Chemistry, University of Maryland at Baltimore, School of Medicine 21201, USA.
Anal Biochem. 1995 May 1;227(1):140-7. doi: 10.1006/abio.1995.1263.
We describe a new class of fluorescence polarization immunoassays based on the luminescence from an asymmetrical Ru-ligand complex. We found that such a complex displays larger polarization values than those of comparable symmetrical complexes and appear to be highly photostable in aqueous solution. We synthesized a conjugatable Ru-ligand complex, which was used to label human serum albumin (HSA) as the antigen. The Ru-ligand complex displays a long decay time near 400 ns when covalently linked to proteins. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format using unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high-molecular-weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2-5 ns decay time of the previously used fluorophores.
我们描述了一类基于不对称钌配体络合物发光的新型荧光偏振免疫分析方法。我们发现，此类络合物比类似的对称络合物显示出更大的偏振值，并且在水溶液中似乎具有高度的光稳定性。我们合成了一种可共轭的钌配体络合物，并用其标记人血清白蛋白（HSA）作为抗原。当与蛋白质共价连接时，钌配体络合物在400 ns附近显示出较长的衰减时间。我们发现，标记的HSA的稳态偏振对抗-HSA的结合敏感，导致偏振增加200%。标记的HSA还以竞争形式使用未标记的HSA作为抗原。时间分辨各向异性衰减表明，在存在抗-HSA的情况下，标记的HSA的相关时间增加，在存在未标记的HSA的情况下，这种效应部分逆转。这些结果证明了金属配体络合物在高分子量分析物荧光偏振免疫分析中的应用潜力。使用此类金属配体络合物能够进行荧光偏振免疫分析，从而绕过了对低分子量抗原的通常限制，这是先前使用的荧光团2-5 ns衰减时间的结果。
