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: R2 = 0.9994, TNF-α: R2 = 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: R2 = 0.9954, TNF-α: R2 = 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.
Highlights
The expression of inflammatory cytokines including interleukin6 (IL-6) and tumor necrosis factor-a (TNF-a) has been associated with acute inflammation and a number of chronic conditions associated with aging, such as cardiovascular disease, diabetes, physical disabilities and cognitive decline [1]
We demonstrated the potential of the sandwich enzymelinked immunosorbant assay (ELISA) on a microchip, with the 1st antibody deposited by piezoelectric inkjet printing for the quantitative analysis of IL-6 and TNF-a which concentrations are very low in the blood of healthy individuals
The luminescence intensities for both IL-6 and TNF-a increased in a concentration-dependent manner (Fig. 2A, Fig. 3A), the luminescence intensities at 2 pg/ml were very weak for both proteins
Summary
The expression of inflammatory cytokines including interleukin (IL-6) and tumor necrosis factor-a (TNF-a) has been associated with acute inflammation and a number of chronic conditions associated with aging, such as cardiovascular disease, diabetes, physical disabilities and cognitive decline [1] Plasma concentrations of these cytokines at reference values are several pg/ml, and they increase in several of the diseases mentioned above. 50 ml aliquots of capture antibody (1st antibody), enzyme conjugated antibody (2nd antibody) to detect antigen, and 50 ml aliquots of antigen solution are introduced into the reaction wells of 96-well microtitration plates [4] This method is time-consuming, for the capture of antigen by the 1st antibody and the determination of the antigen concentration by the 2nd antibody each requires 2 hr or more. The conventional sandwich ELISA for the determination of plasma cytokines is not suitable for rapid diagnosis
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