A novel and sensitive ELISA reveals that the soluble form of IL-13R-alpha2 is not expressed in plasma of healthy or asthmatic subjects.
BACKGROUND
IL-13 plays a key regulatory role in asthmatic responses and immunity to parasitic infection. In vivo, IL-13R-alpha2 is a critical modulator of IL-13 bioactivity. When inducibly expressed on the surface of fibroblasts and other cell types under inflammatory conditions, IL-13R-alpha2 contributes to resolution of IL-13 responses. A soluble form of IL-13R-alpha2 (sIL-13R-alpha2) can be detected in murine circulation, and functions as a regulator of IL-13 bioactivity. In humans, sIL-13R-alpha2 has been more difficult to detect. Recently, novel assay systems have been described to quantitate sIL-13R-alpha2 in human circulation, and revealed unexpectedly high levels of sIL-13R-alpha2 in healthy subjects.


OBJECTIVE
To verify sIL-13R-alpha2 quantitation in human plasma samples under stringent conditions of signal verification and false-positive detection.


METHODS
A standard ELISA protocol was evaluated for specificity using false-positive detection reagents. A more stringent ELISA protocol was developed by optimizing the composition of blocking and dilution buffers.


RESULTS
Using the stringent assay protocol, endogenous sIL-13R-alpha2 was undetectable in plasma samples from a total of 120 asthmatics and 20 healthy subjects, and in bronchoalveolar lavage fluid from 10 asthmatics and eight healthy subjects undergoing allergen challenge.


CONCLUSIONS
These results underscore the necessity to perform rigorous assay controls in the biological matrix to be tested. Because the soluble form could not be demonstrated, our findings question a role for sIL-13R-alpha2 in the regulation of IL-13 bioactivity, and highlight the potentially important contribution of the membrane-bound form of IL-13R-alpha2 in humans.
O'Toole
M
M
Department of Biological Technologies, Wyeth Research, Cambridge, MA 02140, USA.
Legault
H
H
Ramsey
R
R
Wynn
T A
TA
Kasaian
M T
MT
D001249:Asthma / Q000097:blood*; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005189:False Positive Reactions; D006801:Humans; D053663:Interleukin-13 Receptor alpha2 Subunit / Q000096:biosynthesis / Q000097:blood*; D011237:Predictive Value of Tests; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility
D053663:Interleukin-13 Receptor alpha2 Subunit