Rubina A Yu, Dyukova V I, Dementieva E I, Stomakhin A A, Nesmeyanov V A, Grishin E V, Zasedatelev A S
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 ul. Vavilova, 119991 Moscow, Russia.
Anal Biochem. 2005 May 15;340(2):317-29. doi: 10.1016/j.ab.2005.01.042.
Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.
带有固定化蛋白质的三维凝胶基微芯片被用于一系列植物毒素（蓖麻毒素和相思子毒素）和细菌毒素（葡萄球菌肠毒素B、破伤风毒素、白喉毒素以及炭疽致死因子）的定量免疫分析。结果表明，在凝胶微芯片上可以进行不同类型的免疫分析（直接法、竞争法和夹心型）。共聚焦显微镜研究显示，涉及三级抗体 - 抗原 - 抗体复合物形成的抗原 - 抗体相互作用发生在微芯片凝胶元件的整个体积中。使用固定化抗体的微芯片上的夹心分析提供了最高的检测灵敏度（蓖麻毒素为0.1 ng/ml）。用荧光染料标记的抗体、辣根过氧化物酶缀合物或生物素化抗体随后用标记抗生物素蛋白处理作为显色抗体。免疫分析结果直接从微芯片凝胶元件使用荧光、化学发光或基质辅助激光解吸电离质谱记录。带有固定化捕获抗体的凝胶微芯片用于同时分析样品中所有六种生物毒素的存在情况，其灵敏度与任何单一毒素相同。
