Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma.
BACKGROUND
Induced sputum is widely used in asthma research; however, for many mediators, the detection methods have not been validated.


OBJECTIVE
We sought to optimize the method of detection of eotaxin, an important chemokine acting through the CCR3 receptor on eosinophils, basophils, and T(H)2 cells.


METHODS
Induced sputum from normal and asthmatic subjects was processed with dithioerythritol (DTE) or PBS; recovery of eotaxin was assessed by means of ELISA before and after spiking with recombinant eotaxin. Furthermore, the effects of removing DTE by means of ultrafiltration or the addition of protease inhibitors and high-speed centrifugation on endogenous levels and spiking recovery of eotaxin were assessed.


RESULTS
Endogenous eotaxin was undetectable in DTE-processed samples, with a mean of only 30% (SD, 13%) spike recovery. DTE had no effect on the immunoassay capture antibody but dramatically reduced the detection of recombinant eotaxin. Removal of DTE from sputum before immunoassay did not improve detection, although it restored the recovery of a subsequent eotaxin spike. In contrast, PBS-processed sputum resulted in an eotaxin spike recovery of 101% (SD, 20%). Addition of protease inhibitors or high-speed centrifugation had no effect on eotaxin detection. By using this optimized protocol, eotaxin levels in PBS-processed sputum samples were found to be significantly increased in asthmatic sputum (P<.05).


CONCLUSIONS
Measurement of eotaxin by means of immunoassay is adversely affected by DTE, possibly through irreversible denaturation of epitopes, which makes eotaxin undetectable by using the immunoassay antibody. Sputum samples should be processed into PBS for assessment of eotaxin, which is present at increased levels in asthmatic sputum.
Hadjicharalambous
Chrystalleni
C
Department of Respiratory Cell and Molecular Biology, Division of Infection, Inflammation and Repair, University of Southampton School of Medicine, General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom.
Dent
Gordon
G
May
Richard D
RD
Handy
Rachel L C
RL
Anderson
Ian K
IK
Davies
Donna E
DE
Djukanovic
Ratko
R
D000328:Adult; D001249:Asthma / Q000378:metabolism*; D002498:Centrifugation; D054413:Chemokine CCL11; D019742:Chemokines, CC / Q000032:analysis*; D004226:Dithioerythritol / Q000009:adverse effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008875:Middle Aged; D011480:Protease Inhibitors; D013183:Sputum / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000009:adverse effects
C515456:CCL11 protein, human; D054413:Chemokine CCL11; D019742:Chemokines, CC; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D004226:Dithioerythritol