Open AccessA novel flow cytometric assay to quantify soluble CD14 concentration in human serum
Author(s) -
AntalSzalmás Péter,
Szöllősi Ibolya,
Lakos Gabriella,
Kiss Emese,
Csípő István,
Sümegi Andrea,
Sipka Sándor,
van Strijp Jos A.G.,
van Kessel Kok P.M.,
Szegedi Gyula
Publication year - 2001
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/1097-0320(20011001)45:2<115::aid-cyto1153>3.0.co;2-m
Subject(s) - monoclonal antibody , flow cytometry , microbiology and biotechnology , cd14 , fluorescein isothiocyanate , chemistry , serial dilution , antibody , immunology , biology , medicine , fluorescence , pathology , physics , alternative medicine , quantum mechanics
Background 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. Methods 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). Results 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. Conclusions The method is easy, rapid, sensitive, and can be used in the follow‐up of patients suffering from sepsis or chronic inflammatory disorders. Cytometry 45:115–123, 2001. © 2001 Wiley‐Liss, Inc.