title: Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip.
abstract: 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.
authors: Health Research Institute, National Institute of Advanced Industrial Science and Technology AIST, Takamatsu, Japan.
authors: Kaori
authors: K
authors: Abe
authors: Yoshiko
authors: Y
authors: Hashimoto
authors: Shouki
authors: S
authors: Yatsushiro
authors: Shohei
authors: S
authors: Yamamura
authors: Mika
authors: M
authors: Bando
authors: Yuka
authors: Y
authors: Hiroshima
authors: Jun-ichi
authors: J
authors: Kido
authors: Masato
authors: M
authors: Tanaka
authors: Yasuo
authors: Y
authors: Shinohara
authors: Toshihiko
authors: T
authors: Ooie
authors: Yoshinobu
authors: Y
authors: Baba
authors: Masatoshi
authors: M
authors: Kataoka
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015850:Interleukin-6 / Q000097:blood*; D056656:Lab-On-A-Chip Devices; D008163:Luminescent Measurements; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000097:blood*
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha