Quantification of a panel for dry-eye protein biomarkers in tears: A comparative pilot study using standard ELISA and customized microarrays

Abstract

Purpose: This paper examines the tear concentration of cystatin S (CST4), calcyclin (S100A6), calgranulin A (S100A8), and matrix metalloproteinase 9 (MMP9), and the correlation between biomarker expression, clinical parameters, and disease severity in patients suffering from dry eye (DE). A comparison of the results is obtained via ELISA tests and customized antibody microarrays for protein quantification.

Methods: This single-center, observational study recruited 59 participants (45 DE and 14 controls). Clinical evaluation included an Ocular Surface Disease Index (OSDI) questionnaire, a tear osmolarity (OSM) test, the Schirmer test (SCH), tear breakup time (TBUT), fluorescein (FLUO) and lissamine green (LG) corneal staining, and meibomian gland evaluation (MGE). Tear concentrations of CST4, S100A6, S100A8, and MMP9 were measured using standard individual ELISA assays. The levels of CST4, S100A6, and MMP9 were also measured using customized multiplexed antibody microarrays. Correlations between variables were evaluated, and a significance level was p value <0.05.

Results: The quantification of tear protein biomarkers with ELISA showed that the concentration of CST4 was significantly (2.14-fold) reduced in tears of DE patients in comparison with control (CT) subjects (p < 0.001). S100A6 and S100A8 concentrations were significantly higher in the tears of DE patients (1.36- and 2.29-fold; p < 0.001 and 0.025, respectively) in comparison with CT. The MMP9 level was also higher in DE patients (5.83-fold), but not significantly (p = 0.22). The changes in CST4 and S100A6 concentrations were significantly correlated with dry eye disease (DED) severity. Quantification of CST4, S100A6, and MMP9, using antibody microarrays, confirmed the ELISA results. Similar trends were observed: 1.83-fold reduction for CST4 (p value 0.01), 8.63-fold increase for S100A6 (p value <0.001) and 9.67-fold increase for MMP9 (p value 0.94), but with higher sensitivity. The biomarker concentrations were significantly associated with the signs and symptoms related with DED.

Conclusions: S100A6, S100A8, and CST4 diagnostic biomarkers strongly correlate with DED clinical parameters. S100A6 and CST4 are also useful for grading DE severity. The multiplexed antibody microarray technique, used here for tear multi-marker quantification, appears more sensitive than standard ELISA tests.

Figure 1

Workflow diagram of the experimental strategy used in the study. A: Tear samples were collected twice from each volunteer, with a 15 min interval between samplings. The study had two phases (see B and C). B: The first single biomarker quantification, using individual ELISA assays, followed by statistical and correlation analyses. C: The second simultaneous quantification of biomarkers, using multiplexed antibody microarrays. The results obtained using the two techniques were compared. SC: Standard curve.

Figure 2

Dot plots depicting changes in the concentration of CST4, S100A6, S100A8, and MMP9 proteins. A: Comparison of the results for the entire DE group (n = 32) and CT group (n = 14). B: Changes in the DE group categorized by severity status: mild (MILD; n = 11), moderate (MOD; n = 14), and severe (SEV; n = 7), in comparison with the CT group. The (+) indicates the mean and the red line indicates the median. The significance of the results is shown in Table 4.

Figure 3

Correlation of validated tear biomarkers with clinical endpoints. A: Hierarchical clustering of variables. The horizontal axis of the dendrogram represents the distance or dissimilarity between clusters. B: Correlation matrix of biomarker concentrations and clinical parameters. The intense green and intense red represent significant positive and negative correlations, respectively. Significant correlations are indicated in bold (p value <0.05). OSDI: ocular surface disease index; SCH: Schirmer test; MH: meniscus height; OSM: osmolarity; FLUO: fluorescein staining; LG: lissamine green; TBUT: tear breakup time; MGE: meibomian gland expression; MGQ: meibomian gland quality.

Figure 4

Comparison of mean biomarker concentrations obtained with ELISA and with antibody microarrays in the DE and CT groups. A: Comparison of the concentrations of CST4, S100A6, and MMP9 biomarkers in control (CT; n = 14) and dry eye (DE; n = 32) groups measured using the standard individual ELISA assays and customized multiplexed antibody microarrays. Concentration is expressed in ng/ml. The bars represent a comparison of mean biomarker concentration in the DE group relative to the CT group. Error bars show standard deviation, and * indicates statistical significance relative to control (p value <0.05). B: Regression analyses of the results obtained with ELISA and antibody microarray technology. In all three cases, the p values ​​associated with the linear regressions were lower than 0.001.

Figure 5

Comparison of the mean concentrations of biomarkers CST4 (A), S100A6 (B), and MMP9 (C) in control (CT; n = 14) and dry eye (DE) groups according to the severity (mild, n = 11; moderate, n = 14; and severe, n = 7), measured using standard individual ELISA assays and customized multiplexed antibody microarrays. The concentration is expressed in ng/ml. The insert in the S100A6 plot shows the details of the results obtained using ELISA assays. Error bars show standard deviation, and * indicates statistical significance relative to the corresponding control group (p value <0.05).