Determination of ELISA reproducibility to detect protein markers in exhaled breath condensate

Exhaled breath condensate (EBC) is a representative sample from the lungs that may be used to detect different markers, but the reproducibility of these determinations is unknown over time. The aim of this paper is to assess the reproducibility of protein marker determination in EBC using samples collected at two different time points. EBC and blood were collected from 16 healthy subjects, smokers and non-smokers by using the ECoScreen device. EBC was collected on two separate occasions within ten days. Enzyme-linked immunosorbent assay (ELISA) was performed to measure angiogenesis and hypoxia markers. Blood and EBC samples were analyzed by ELISA to detect angiogenesis markers: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin. A hypoxia marker, the anhydrase IX, was also determined. Biomarker concentration was higher in plasma samples compared to EBC. bFGF determination was higher in women (39.47 ± 3.914 versus 27.15 ± 3.145; p < 0.05). There were no significant differences among the averages of detection for any of the markers. The Bland-Altman method showed that the average of the differences or biases in EBC for every biomarker was close to zero, indicating a good reproducibility of the measurements. Nevertheless, the VEGF showed wide limits of agreement. EBC is suitable to detect biomarkers by ELISA and the measurements are reproducible over time. Nevertheless, some factors such as sex should be taken into account when analyzing the results.