Health Research Institute, National Institute of Advanced Industrial Science and Technology AIST, Takamatsu, Japan.
PLoS One. 2013;8(1):e53620. doi: 10.1371/journal.pone.0053620. Epub 2013 Jan 9.
Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R(2) = 0.9994, TNF-α: R(2) = 0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R(2) = 0.9954, TNF-α: R(2) = 0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.
夹心酶联免疫吸附试验（ELISA）使用 96 孔板常用于临床诊断，但时间和样品消耗大。为了克服这些缺点，我们在微芯片上进行了夹心 ELISA。微芯片由环状烯烃共聚物制成，有 4 个直的微通道。为了构建白细胞介素 6（IL-6）或肿瘤坏死因子-α（TNF-α）的夹心 ELISA，我们使用压电喷墨打印系统将第一抗 IL-6 抗体或第一抗 TNF-α 抗体沉积并固定在每个微通道的表面上。在将 2 μl 样品注入微通道并孵育 20 分钟后，注入 2 μl 生物素化的第二抗体用于任一抗原，并孵育 10 分钟。然后注入 2 μl 亲和素-辣根过氧化物酶；孵育 5 分钟后，注入过氧化物酶的底物，测量发光强度。在 0 至 32 pg/ml 的浓度范围内获得了与发光强度之间的校准曲线（IL-6：R²=0.9994，TNF-α：R²=0.9977），每种蛋白质的检测限分别为 0.28 pg/ml 和 0.46 pg/ml。将从微芯片数据估计的 5 个个体的血液 IL-6 和 TNF-α 浓度与通过常规方法获得的结果进行比较，根据线性回归分析观察到两种方法之间的良好相关性（IL-6：R²=0.9954，TNF-α：R²=0.9928）。所提出的用于测定血液 IL-6 和 TNF-α 浓度的测定方法的重现性与使用 96 孔板获得的结果相当。通过将每种第一抗体沉积并固定在单独的微通道表面上，可以同时检测血液 IL-6 和 TNF-α。该测定法使我们能够准确、灵敏、节省时间地同时测定血液 IL-6 和 TNF-α，并且样品和试剂的消耗低，将适用于临床诊断。
