Analytical Sciences

Highly Sensitive Determination of Plasma Cytokines by Time-Resolved Fluoroimmunoassay; Effect of Bicycle Exercise on Plasma Level of Interleukin-1alpha (IL-1alpha), Tumor Necrosis Factor alpha (TNFalpha), and Interferon gamma (IFNgamma)

Hiroko KIMURA,*¹ Masatoshi SUZUI,*² Fumiko NAGAO,*³ and Kazuko MATSUMOTO*⁴

*1 Department of Forensic Medicine, Juntendo University School of Medicine, Hongo, Tokyo 113-8421, Japan
*2 School of Business Administration, Health Science, Meiji University, Suginami, Tokyo 168-8555, Japan
*3 Department of Immunology, Juntendo University School of Medicine, Hongo, Tokyo 113-8421, Japan
*4 Department of Chemistry, Waseda University, Okubo, Shinjuku, Tokyo 169-8555, Japan

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNFalpha) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu³⁺ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1alpha, TNFalpha, and interferon gamma (IFNgamma) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1alpha, TNFalpha, and IFN gamma were determined. In the case of incremental exercise, IL-1alpha increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN gamma increased in recovery period, 2 h after the light-exercise. TNFalpha level was significantly higher in a heavy-exercise. The concentration of IL-1alpha peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

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