Wang Hailin, Lu Meiling, Weinfeld Michael, Le Chris X
Environmental Health Sciences Program, Department of Public Health Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.
Anal Chem. 2003 Jan 15;75(2):247-54. doi: 10.1021/ac026204a.
The stability of antibody and formation of immunocomplexes are essential to high-sensitivity capillary electrophoresis immunoassays (CEIA). However, little attention has been paid to enhancing or maintaining immunocomplex formation and antibody stability to improve the performance of CEIA. We report here the use of nonspecific proteins, such as bovine serum albumin (BSA) and rabbit immunoglobulin (rIgG), to enhance immunocomplex formation and to stabilize antibodies and immunocomplexes for immunoassays. Complexes between DNA adducts of benzo[a]pyrenediol epoxide (BPDE) and their antibodies were examined using capillary electrophoresis with laser-induced fluorescence detection (CE-HF). A tetramethylrhodamine (TMR)-labeled single-stranded oligonucleotide (16-mer) containing a single BPDE adduct was used as a fluorescent probe to study its immunocomplexes with a monoclonal antibody (8E11). To examine the formation of larger complexes, a TMR-labeled secondary antibody (anti-mouse), a primary antibody (mouse monoclonal antibody 5D11), and BPDE adducts in cellular DNA were used. We demonstrate that the use of nonspecific proteins stabilized the antibody and greatly enhanced the formation and stability of the immunocomplexes, resulting in substantial improvements in the detection limit (10-fold) and the reproducibility of the analysis. Another advantageous consequence of the stabilization was a 150-fold reduction of the concentration of the antibody needed for the immunoassay, resulting in reduced background and cost. We successfully applied this technique to the determination of DNA adducts of BPDE using a competitive immunoassay. The results from both small complexes (between a primary antibody and an oligonucleotide) and larger complexes (among a secondary antibody, a primary antibody, and cellular DNA) indicate that the technique can be extended to other immunoassays. We suggest that nonspecific proteins may assist the formation and stabilization of antibody-antigen complexes by maintaining the correct conformation of the antibody and antigen for optimum binding.
抗体的稳定性和免疫复合物的形成对于高灵敏度毛细管电泳免疫分析（CEIA）至关重要。然而，在增强或维持免疫复合物形成以及抗体稳定性以提高CEIA性能方面，人们关注较少。我们在此报告使用非特异性蛋白质，如牛血清白蛋白（BSA）和兔免疫球蛋白（rIgG），来增强免疫复合物形成，并稳定用于免疫分析的抗体和免疫复合物。使用毛细管电泳结合激光诱导荧光检测（CE-HF）来检测苯并[a]芘二醇环氧化物（BPDE）的DNA加合物与其抗体之间的复合物。一种含有单个BPDE加合物的四甲基罗丹明（TMR）标记的单链寡核苷酸（16聚体）被用作荧光探针，以研究其与单克隆抗体（8E11）的免疫复合物。为了检测更大复合物的形成，使用了TMR标记的二抗（抗小鼠）、一抗（小鼠单克隆抗体5D11）以及细胞DNA中的BPDE加合物。我们证明，使用非特异性蛋白质可稳定抗体，并极大地增强免疫复合物的形成和稳定性，从而使检测限大幅提高（10倍），并改善分析的重现性。稳定化的另一个有利结果是免疫分析所需抗体浓度降低了150倍，从而降低了背景和成本。我们成功地将该技术应用于使用竞争性免疫分析测定BPDE的DNA加合物。来自小复合物（一抗与寡核苷酸之间）和大复合物（二抗、一抗与细胞DNA之间）的结果均表明，该技术可扩展至其他免疫分析。我们认为，非特异性蛋白质可能通过维持抗体和抗原的正确构象以实现最佳结合，从而协助抗体-抗原复合物的形成和稳定。
