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Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip.
PLoS One. 2013; 8(1):e53620.Plos

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+Show Affiliations

Health Research Institute, National Institute of Advanced Industrial Science and Technology AIST, Takamatsu, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23326472

Citation

Abe, Kaori, et al. "Simultaneous Immunoassay Analysis of Plasma IL-6 and TNF-α On a Microchip." PloS One, vol. 8, no. 1, 2013, pp. e53620.
Abe K, Hashimoto Y, Yatsushiro S, et al. Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip. PLoS ONE. 2013;8(1):e53620.
Abe, K., Hashimoto, Y., Yatsushiro, S., Yamamura, S., Bando, M., Hiroshima, Y., Kido, J., Tanaka, M., Shinohara, Y., Ooie, T., Baba, Y., & Kataoka, M. (2013). Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip. PloS One, 8(1), e53620. https://doi.org/10.1371/journal.pone.0053620
Abe K, et al. Simultaneous Immunoassay Analysis of Plasma IL-6 and TNF-α On a Microchip. PLoS ONE. 2013;8(1):e53620. PubMed PMID: 23326472.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip. AU - Abe,Kaori, AU - Hashimoto,Yoshiko, AU - Yatsushiro,Shouki, AU - Yamamura,Shohei, AU - Bando,Mika, AU - Hiroshima,Yuka, AU - Kido,Jun-ichi, AU - Tanaka,Masato, AU - Shinohara,Yasuo, AU - Ooie,Toshihiko, AU - Baba,Yoshinobu, AU - Kataoka,Masatoshi, Y1 - 2013/01/09/ PY - 2012/10/16/received PY - 2012/11/30/accepted PY - 2013/1/18/entrez PY - 2013/1/18/pubmed PY - 2013/7/3/medline SP - e53620 EP - e53620 JF - PloS one JO - PLoS ONE VL - 8 IS - 1 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/23326472/Simultaneous_immunoassay_analysis_of_plasma_IL_6_and_TNF_α_on_a_microchip_ L2 - http://dx.plos.org/10.1371/journal.pone.0053620 DB - PRIME DP - Unbound Medicine ER -