Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin, China.
Anal Chem. 2012 Mar 20;84(6):2708-12. doi: 10.1021/ac202959d. Epub 2012 Feb 28.
A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.
基于磁珠上化学发光共振能量转移（CRET）的竞争免疫分析用于检测人免疫球蛋白 G（IgG）。在该方案中，羧基修饰的磁珠与辣根过氧化物酶（HRP）标记的山羊抗人 IgG（HRP-anti-IgG）偶联，并与有限量的荧光素异硫氰酸酯（FITC）标记的人 IgG 孵育以将抗体-抗原免疫复合物固定在磁珠表面上，然后进一步与靶标分析物（人 IgG）进行竞争性免疫反应，并通过磁性分离去除上清液。然后加入化学发光（CL）缓冲液（含有鲁米诺和 H(2)O(2)），并立即在磁珠表面的免疫复合物中发生供体鲁米诺到受体 FITC 的 CRET。本方案用于人 IgG 的竞争免疫分析，在 0.2-4.0 nM 范围内 CL 强度比（R = I(425)/I(525)）与 IgG 浓度之间呈现线性关系，相关系数为 0.9965。回归方程表示为 R = 1.9871C + 2.4616，检测限为 2.9×10(-11) M。本方法成功地用于检测人血清中的 IgG。结果表明，本方案在 CRET 在免疫分析中的应用中具有很大的应用潜力。通过使用相应的抗原和各自的抗体，也可以开发用于检测其他抗原-抗体免疫复合物的方法。
