Hall M, Kazakova I, Yao Y M
Pharmaceutical Discovery Division, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, USA.
Anal Biochem. 1999 Aug 1;272(2):165-70. doi: 10.1006/abio.1999.4155.
The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.
本文描述了聚苯乙烯荧光微球在96孔板直接检测（非酶放大）异相平衡“夹心”免疫分析中作为灵敏标记物的应用。以小鼠IgG作为模型抗原，荧光颗粒标记物比相应的可溶性报告分子更灵敏。在多参数优化分析中，颗粒报告分子检测小鼠IgG的检测限为0.2 ng/ml（7.6×10⁸个抗原/ml），可溶性报告分子的检测限为50 ng/ml（1.9×10¹¹个抗原/ml）。使用颗粒标记物的分析灵敏度取决于捕获抗体和报告抗体的表面密度以及报告微球的浓度。通过将预先形成的报告抗体/荧光微球复合物添加到孔表面捕获的抗原上，而不是依次添加报告抗体和荧光微球，灵敏度得到了提高。当报告微球浓度为1.4×10⁹个/ml时，颗粒报告分子与捕获抗原的最大（平衡）结合在20小时后出现。因此，颗粒荧光标记物在直接检测免疫分析中提供了高灵敏度。
