In brief, a monoclonal anti-IL-4 antibody-coated microtiter plate was incubated with human IL-4 standards or sera in a total volume of 0.1 ml at 20°C for 16 h. Human IL-4 standards were diluted with 10 mmol/L phosphate buffer (PBS), at a pH of 6.8, containing 10 mg/L of BSA, 0.2 mol/L NaCl, and 0.1 g/L NaN3. Following removal of the incubation mixture, the plate was washed three times with PBS-Tween buffer and incubated with affinity-purified anti-IL-4 rabbit IgG in 0.1 ml at 20°C for 3.5 h. The incubation mixture was removed again and the plate was washed three times and incubated with affinity-purified antirabbit IgG F(ab’)2-ALP at 20°C for 2 h. After one more removal of the incubation mixture, the plate was washed four times as described above. Bound ALP activity was assayed at 25°C for 60 min with AMPPD (with Emerald Amplifier), and relative chemiluminescent intensity was measured with a microplate luminometer. This system was capable of detecting 0.03 to 50 pg/ml of human IL-4.
The serum level of sFceRII was determined with sandwich ELISA. Antibodies against FceRII were purchased (Yamasa Shoyu Co, Ltd, Japan) and conjugated with peroxidase. The antibody characteristics have already been described. The assay was performed on a microtiter plate as above. Values were expressed as unit per milliliter of serum. Minimum assay sensitivity was 6.0 U/ml. canadianneighborpharmacy.com
Means of the asthmatic group and control group were compared by the unpaired Student’s t test. Differences associated with probability of p < 0.05 were considered significant. Correlation coefficients and statistical significance were determined by Pearson’s linear regression analysis. All data were expressed as means ± SEM. Serum sIL-2R, IL-4, and sFceRII in asthmatics significantly increased, compared with the controls (sIL-2R: 459.0130.4 U/ml vs 251.5 ±10.0 U/ml, p< 0.001) (IL-4: 1.89 ±0.13 pg/ml vs 1.08 ±0.15 pg/ml, p< 0.001) (sFceRII: 313.2 ±18.8 U/ml vs 228.8 ± 6.7 U/ml, p < 0.001) (Fig 1). Serum levels of these three parameters did not depend on age, sex, treatment, FEVj percent predicted, and histamine PC20 (data not shown).
Mutual Correlations Among sIL-2R, IL-4, and sFceRII in Asthmatics
Serum sIL-2R showed a weak but significant correlation with sFceRII (Fig 2) (r = 0.46, p < 0.001), but did not correlate with IL-4 (r = 0.03). Also, there was no correlation between IL-4 and sFceRII (r = 0.05).
Comparison of slL-2Rt IL-4, and sFceRII in the Two Asthmatic Groups Based on Atopic Status
Atopic asthmatics were characterized by a higher total IgE and a lower mean age than the nonatopic group (p < 0.001, p < 0.01, respectively).
Figure 1. Serum sIL-2R, IL-4 and sFceRII in asthmatics and controls. Geometric means ± sem. The levels of all parameters in asthmatics significantly increased as compared with those in the controls.
Figure 2. Correlation between slL-2R and sFceRII in asthmatics. Significant correlation was noted.