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

The CC chemokine eotaxin is a potent eosinophil-selective chemoattractant, and it is thought that the function of eotaxin is closely related to the re cruitment of eosinophils in certain inflammatory reactions. In order to lea rn more about the biological role of this molecule, we have developed a new sandwich ELISA method to measure human eotaxin using two monoclonal antibo dies and purified recombinant eotaxin as a standard. The minimal detectable concentration of eotaxin in this assay was 1.5 pg/ml, and the working rang e was 3.1-200 pg/ml with low CVs (<10%). Both within- and between-run preci sion levels were less than 6.7% of the CVs. The dilution curves of two seru m and two spiked plasma samples showed good linearity and the recovery rang e was 92.8-103.3%. No cross-reactivity was found with other similar chemoki nes, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin-2 and RANTES. This assay was sensi tive enough to measure the circulating eotaxin levels of healthy volunteers . However, the eotaxin levels in serum samples (mean +/- SD; 68.6 +/- 13.4 pg/ml, n = 15) were significantly higher than those in matched plasma sampl es (19.2 +/- 5.4 pg/ml) separated from blood collected in tubes containing EDTA. Kinetic studies revealed that the eotaxin levels in serum markedly in creased depending on the elapsed time before separation from blood cells, b ut such changes in EDTA-plasma were negligible up to 4 h at 25 degrees C. O ur new ELISA is an accurate and useful method for quantifying human eotaxin in blood and demonstrates that the process of preparing blood samples affe cts the measurement of the eotaxin levels. (C) 1999 Elsevier Science B.V. A ll rights reserved.