A novel flow cytometric assay to quantify soluble CD14 concentration in human serum.
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.
Antal-Szalmás
P
P
3rd Department of Internal Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Szöllosi
I
I
Lakos
G
G
Kiss
E
E
Csípo
I
I
Sümegi
A
A
Sipka
S
S
van Strijp
J A
JA
van Kessel
K P
KP
Szegedi
G
G
D000328:Adult; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000097:blood*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000737:chemistry; D012372:ROC Curve; D012680:Sensitivity and Specificity
D018950:Lipopolysaccharide Receptors