A novel flow cytometric assay to quantify soluble CD14 concentration in human serum.

Abstract

CD14, the major lipopolysaccharide (LPS)-binding protein of myeloid cells, is found as a soluble molecule in human serum. Recent data describe the presence of elevated soluble CD14 (sCD14) concentration in various disorders, confirming disease activity. A novel, easy, and rapid flow cytometric assay was developed to measure sCD14 levels in serum. The assay is based on the competition between membrane-expressed CD14 of isolated monocytes from healthy volunteers and sCD14 in the sample sera for binding to anti-CD14 monoclonal antibodies (mAb; 26ic or 60bca). The amount of cell-associated mAb is determined with a fluorescein isothiocyanate (FITC)-labeled anti-mouse conjugate and flow cytometry. The fluorescence signal is inversely proportional with the amount of serum sCD14. Using dilutions of a standard serum, the concentration of sCD14 in the samples is calculated and compared with results obtained by a commercial sCD14 enzyme-linked immunosorbent assay (ELISA). After optimization, the assay showed log-log linearity of 122.1-984.7 ng/ml sCD14 using mAb 26ic and 29.5-246.2 ng/ml sCD14 using mAb 60bca. It revealed similar results as the ELISA (mAb 26ic: r = 0.88, mAb 60bca: r = 0.92) and provided significantly elevated sCD14 levels in systemic lupus erythematosus patients compared with controls (26ic: 2,213 versus 1,676 ng/ml, P < 0.002; 60bca: 2,625 versus 1,907 ng/ml, P < 0.0002). Receiver operating characteristic curve analysis suggested a reasonable diagnostic efficacy of sCD14 quantification in this autoimmune disease. The method is easy, rapid, sensitive, and can be used in the follow-up of patients suffering from sepsis or chronic inflammatory disorders.