Technical problems with protein extraction of chemokines featuring RANTES.
Chemokines are known to be one of the sources for eosinophilic tissue infiltration in eosinophilic inflammation. Detection of beta-chemokines such as RANTES was possible in nasal tissue with or without eosinophilic infiltration. The concentration of chemokines which has been measured in the same tissue differs often in the literature. Aim of this study was to compare the different techniques of protein extraction and help to understand and interpret the investigation on RANTES secretion. Tissue of nasal polyps, inferior and middle turbinate was cut into halves and every half on its own pulverized using liquid nitrogen. The protein extraction was performed either with citric acid solution (pH 2.5) or phosphate buffered saline (PBS). The samples were then lyophilized. The concentration of RANTES was measured by a specific double sandwich ELISA. Using the citric acid technique the average concentration of RANTES in middle turbinates was 1.3 ng/mg, in inferior turbinates 1.6 ng/mg and in polyps 2.6 ng/mg tissue, using the PBS technique respectively 0.6 ng/mg, 0.5 ng/mg and 0.8 ng/mg tissue. Our data revealed a mismatch of 3.3:1 for polyps (citric acid: PBS), 3.2:1 for inferior and 2.2:1 for middle turbinates, respectively. Consequent comparison between the results of different techniques was not possible. Of special interest was also the fact that different techniques had different efficiencies of protein extraction in different tissues. Present statements on RANTES concentrations as a prognostic factor in nasal tissues need a technically careful standardization as far as this study shows.
Maune
S
S
Dept. of Otorhinolaryngology, Head and Neck Surgery, University of Kiel, F.R.G.
Meyer
J E
JE
Spautz
B
B
Sticherling
M
M
Schröder
J M
JM
D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism; D012220:Rhinitis / Q000378:metabolism*; D012852:Sinusitis / Q000378:metabolism*; D014420:Turbinates / Q000166:cytology / Q000378:metabolism
D018946:Chemokine CCL5