Development of a sensitive enzyme-linked immunosorbent assay for eotaxin and measurement of its levels in human blood

Abstract The CC chemokine eotaxin is a potent eosinophil-selective chemoattractant, and it is thought that the function of eotaxin is closely related to the recruitment of eosinophils in certain inflammatory reactions. In order to learn more about the biological role of this molecule, we have developed a new sandwich ELISA method to measure human eotaxin using two monoclonal antibodies and purified recombinant eotaxin as a standard. The minimal detectable concentration of eotaxin in this assay was 1.5 pg/ml, and the working range was 3.1–200 pg/ml with low CVs ( n =15) were significantly higher than those in matched plasma samples (19.2±5.4 pg/ml) separated from blood collected in tubes containing EDTA. Kinetic studies revealed that the eotaxin levels in serum markedly increased depending on the elapsed time before separation from blood cells, but such changes in EDTA-plasma were negligible up to 4 h at 25°C. Our new ELISA is an accurate and useful method for quantifying human eotaxin in blood and demonstrates that the process of preparing blood samples affects the measurement of the eotaxin levels.