Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

Citation: de la Fuente, M.; Rodríguez-Agirretxe, I.; Vecino, E.; Astigarraga, E.; Acera, A.; Barreda-Gómez, G. Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays. Int. J. Mol. Sci. 2022,23, 5639. https://doi.org/ 10.3390/ijms23105639 Academic Editors: Phaedra Eleftheriou and Athina Geronikaki Received: 24 March 2022 Accepted: 16 May 2022 Published: 18 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays Miguel de la Fuente 1,2 , Iñaki Rodríguez-Agirretxe 3, Elena Vecino 2,4 , Egoitz Astigarraga 1, Arantxa Acera 2,5,* and Gabriel Barreda-Gómez 1,* 1Department of Research and Development, IMG Pharma Biotech S.L., 48160 Derio, Spain; [email protected] (M.d.l.F.); [email protected] (E.A.) 2Experimental Ophthalmo-Biology Group (GOBE), Department of Cell Biology and Histology, University of the Basque Country UPV/EHU, 48940 Leioa, Spain; [email protected] 3Department of Ophthalmology, Donostia University Hospital, 20014 San Sebastian, Spain; [email protected] 4Begiker-Ophthalmology Research Group, BioCruces Health Research Institute, 48903 Barakaldo, Spain 5IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain *Correspondence: [email protected] (A.A.); [email protected] (G.B.-G.); Tel.: +34-946-018-024 (A.A.); Tel.: +34-944-316-577 (G.B.-G.) Abstract: Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface. Keywords: tear MMP-9; enzyme biomarker; diagnosis; monitoring; antibody microarray; ocular inflammation; glaucoma; point of care; in vitro diagnostics 1. Introduction Biomarkers can be defined as biological analytes by which a particular pathological or physiological process can be identified or characterized [ 1 ]. They allow a more precise diagnosis and the monitoring of pathologies and conditions. A biomarker can determine if the patient has a particular medical state, the different subtypes of the pathology if applicable, and the best treatment indicated, improving the monitoring of the therapy response, the diagnosis, and the progression [2]. Among all types of biomarkers, enzymes are gaining importance in many pathologies [ 3 , 4 ]. Enzymes are chemical catalysts that help organisms conduct essential biochemical reactions. Deficiency, malfunction, reduced/increased activity, or overexpression of enzymes and their inhibitors can cause a variety of clinical conditions [ 5 ]. Consequently, the study of enzymes and their inhibitors is cardinal for understanding disease pathophysiology and developing not only therapeutic options but also diagnostic and monitoring strategies, as enzymes are powerful markers of disease [ 5 , 6 ]. In this regard, detection and Int. J. Mol. Sci. 2022,23, 5639. https://doi.org/10.3390/ijms23105639 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 5639 2 of 16 quantification of enzymes in biological fluids is an interesting field of research, as it can lead to improvements in pathology prognosis and patient life. One of the main processes in which enzymes participate is inflammation, a fundamental mechanism for maintenance of body homeostasis versus infections and injuries. Novel published research has established a relationship between systemic inflammation and several highly prevalent pathologies, such as cancer [ 7 ] and neurodegenerative [ 8 ], autoimmune [ 9 ], cardiovascular [ 10 ], and metabolic diseases [ 11 ]. In addition, many ocular pathologies such as Sjogren’s syndrome [ 12 ], or keratoconjunctivitis sicca, commonly named as dry eye (DE) [ 13 ], have also been correlated with inflammation. Furthermore, antimicrobial preservative compounds such as quaternary ammonium benzalkonium chloride (BAK), used in antiglaucoma eye drop treatments, have been associated with chronic ocular inflammation [ 14 , 15 ]. Many clinical symptoms of chronic ocular inflammation have been reported in patients under long-term antiglaucoma treatment [ 16 ]. It has been determined that BAK acts at different levels of the cell machinery, interacting with cell membranes and receptors. It affects conjunctival epithelial cells and provokes ocular inflammation signs and symptoms such as loss of goblet cells, conjunctival squamous metaplasia and apoptosis, disruption of the corneal epithelium barrier, and damage to deeper ocular tissues [ 16 ]. These toxic effects trigger inflammation pathways that precipitate the overexpression of certain enzymes. Taking this into account, enzymes can be used as biomarkers, either for diagnosis or for monitoring the response to a treatment and evaluating the adverse and toxic effects of the therapy. Matrix metalloproteinases (MMPs) are a family of enzymes that play important roles in inflammatory processes [ 17 , 18 ]. MMP-9, also called gelatinase B, is a zinc and calcium ion-dependent enzyme that is involved in tissue remodeling by degrading types IV and V collagen of the extracellular matrix (ECM) in physiological processes such as wound healing and bone growth [ 19 , 20 ]. This enzyme plays an important role and is upregulated in inflammatory pathologies, arthritis, cardiovascular and pulmonary diseases, as well as in cancer [ 18 ]. MMP-9, along with other MMPs, is upregulated during inflammation in different tissues and fluids such as serum, saliva, synovial liquid, or tear, becoming an interesting enzyme biomarker. Thus, detection and quantification of MMP-9 in non-invasive fluids is a promising approach for inflammation prevention, diagnosis, and disease or treatment monitoring. Concretely, MMP-9 has been also extensively studied as a biomarker of inflammation in tear samples [ 21 – 25 ]; this biomarker is highly overexpressed in different diseases associated with ocular inflammation and in ocular surface pathologies [ 22 , 26 , 27 ]. In the corneal epithelium, both TGFβ and IL-1 cytokines, key players in the regulation of inflammatory processes, stimulate MMP-9 overexpression [28]. Currently, the diagnosis of the main ocular surface inflammation pathologies is mostly subjective and is based on the knowledge of the ophthalmologist and the signs and symptoms of the patients [ 29 , 30 ]. However, the discovery and use of biomarkers, such as MMP-9, have opened new lines of research aiming to develop new diagnosis tools [ 31 ]. These biomarkers are extremely useful in the clinic because they reflect the pathological state of the patient, as well as the evolution of the disease in molecular terms; thus, they can be used, not only for diagnosis but also for evaluating the prognosis and for monitoring the progression of the pathology and the response to treatments. Various studies have validated tear MMP-9 as one of the main biomarkers for ocular inflammation diseases [ 23 ]. Different commercial diagnosis point of care (PoC) and in vitro diagnostics (IVDs) tests have been developed for evaluation of tear MMP-9, such as InflammaDry [ 24 , 27 ]; however, these types of tests have the main drawback of giving only a positive/negative result, not allowing the quantification of the biomarker, nor a precise evaluation of the pathological status of the patient, nor the monitoring of the disease, due to their variability [ 32 ]. Additionally, this hampers the correlation between symptoms and biomarker concentration, precluding the stratified diagnosis of patients. Alternatively, microarray technology can be applied as a platform for biomarker-based diagnostics or monitoring. Cell membranes, whole cells, antibodies, enzymes, nucleic acids, Int. J. Mol. Sci. 2022,23, 5639 3 of 16 and other proteins can be immobilized on diverse surfaces using microarray technology without losing their functional structure. As a result, they are used in immunochemistry, autoradiography, radioligand and binding investigations, mitochondrial toxicity assays, as well as other techniques such as colorimetry and mass spectrometry [ 33 – 37 ]. Microarrays allow the reduction of the number of samples, medications, chemicals, and residues. Among them, antibody microarrays (AbMAs) are used similarly as a miniaturized enzymelinked immunosorbent assay (ELISA) for the detection of analytes. However, AbMAs show higher sensitivity for the identification of biomarkers than traditional ELISA, demonstrating their improvements in clinical situations when taking also into account the previously mentioned advantages [ 25 ]. Currently, AbMAs are widely used for disease diagnosis in diverse pathologies such as cancer [38], or ocular conditions [39], among others [40,41]. Hence, the aim of this work was to develop an AbMA test for ocular inflammation detection by quantifying tear MMP-9 biomarker (Figure 1). For this purpose, antibodies against human MMP-9 were immobilized over glass slides where the sample was incubated and the biomarker was captured. Then, the biomarker was detected using a labeled antibody cocktail that produced a fluorescent intensity signal directly proportional to the concentration of MMP-9 in the sample. For the validation of the test, eight non-pathological tear samples were used. Enzyme MMP-9 biomarker concentration was confirmed using conventional ELISA as the gold standard to characterize the samples and assess the reliability of the test. Subsequently, tear samples from 20 individuals clinically diagnosed with ocular inflammation were assayed. Using a calibration line, protein biomarker presence was quantified in each of the samples employing AbMAs, early validating the developed technique as an MMP-9 inflammation-related detection tool. Int. J. Mol. Sci. 2022,23, 5639 4 of 16 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 18 Figure 1. Detection and quantification of MMP-9 enzyme inflammation biomarker in human tear samples using AbMAs. First, the selected antibodies were immobilized onto glass slides that were incubated with the sample. Then, MMP-9 was captured by the mentioned antibody and detected with a labeled antibody cocktail. Finally, the intensity of the signal was quantified and the data acquired, allowing the analysis of the MMP-9 biomarker in the samples. Figure 1. Detection and quantification of MMP-9 enzyme inflammation biomarker in human tear samples using AbMAs. First, the selected antibodies were immobilized onto glass slides that were incubated with the sample. Then, MMP-9 was captured by the mentioned antibody and detected with a labeled antibody cocktail. Finally, the intensity of the signal was quantified and the data acquired, allowing the analysis of the MMP-9 biomarker in the samples. Int. J. Mol. Sci. 2022,23, 5639 5 of 16 2. Results 2.1. Subjects A cohort of samples from both healthy controls and patients suffering ocular inflammation was used to validate customized AbMAs as an MMP-9 quantification assay for ocular inflammation evaluation in human tear fluid. Firstly, tear samples were obtained from volunteers as detailed in the Materials and Methods Section. Tear samples were divided into two groups: healthy controls, named as HC, and patients, named as P. The second one was composed of individuals suffering ocular inflammation due to different pathological conditions such as cataracts, glaucoma, meibomian gland dysfunction (MGD), allergy, or DE. Patients were evaluated using a Schirmer’s test; normal values were considered ≥ 10 mm wetting of the paper after 5 min, whereas tear deficiency values were ≤ 5 mm. All patients, except from P 9, P 11, and P 18, presented tear deficiency. Glaucoma patients were all under prostaglandin eye drop treatment; these drugs were preserved with BAK. P 9 was under two different BAK-preserved prostaglandin analogue treatments. No Schirmer’s test was performed for healthy volunteers to avoid the intervention since they did not present clinical conditions. In addition, the gender and age of the patients were detailed (Table 1). Table 1. Baseline characteristics of the patients and healthy control individuals. The healthy controls were collected from volunteers without any ocular pathology diagnosed. Tear Sample Group Age Gender Conditions Schirmer’s Test (mm) HC 1 Healthy Control 25 Male n/a n/a HC 2 Healthy Control 26 Female n/a n/a HC 3 Healthy Control 30 Female n/a n/a HC 4 Healthy Control 23 Female n/a n/a HC 5 Healthy Control 40 Male n/a n/a HC 6 Healthy Control 23 Female n/a n/a HC 7 Healthy Control 24 Female n/a n/a HC 8 Healthy Control 29 Female n/a n/a P 1 Patient 79 Female Cataracts 5 P 2 Patient 73 Female Cataracts 5 P 3 Patient 66 Female Cataracts 3 P 4 Patient 81 Female Cataracts 5 P 5 Patient 89 Female Cataracts 2 P 6 Patient 62 Female Cataracts 0 P 7 Patient 70 Male Cataracts 1 P 8 Patient 73 Female Cataracts 3 P 9 Patient 68 Female Glaucoma 6 P 10 Patient 60 Male Glaucoma 5 P 11 Patient 75 Female Glaucoma 7 P 12 Patient 70 Female Glaucoma 4 P 13 Patient 82 Male Glaucoma 5 P 14 Patient 82 Male Glaucoma 5 P 15 Patient 52 Male MGD 5 P 16 Patient 49 Female Allergy 4 P 17 Patient 29 Female DE 5 P 18 Patient 30 Female DE 6 P 19 Patient 49 Female Allergy 5 P 20 Patient 38 Female MGD + DE 3 2.2. Antibody Microarray Validation The eight samples from the healthy volunteers were characterized using an antihuman MMP-9 ELISA kit. In order to assess the reliability of this technique, in contrast with the gold standard, the obtained values were compared with the quantification of tear MMP-9 using the developed AbMAs. When comparing the concentration of MMP-9 obtained with each technique, similar results were obtained (Figure 2). Additionally, a simple Int. J. Mol. Sci. 2022,23, 5639 6 of 16 bivariate correlation was calculated, setting the significance at α = 0.05 using a two-tailed test. A Pearson correlation coefficient (r) of 0.9918 was obtained with a significance of (****), p-value < 0.0001. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 18 2.2. Antibody Microarray Validation The eight samples from the healthy volunteers were characterized using an antihuman MMP-9 ELISA kit. In order to assess the reliability of this technique, in contrast with the gold standard, the obtained values were compared with the quantification of tear MMP-9 using the developed AbMAs. When comparing the concentration of MMP-9 obtained with each technique, similar results were obtained (Figure 2). Additionally, a simple bivariate correlation was calculated, setting the significance at α = 0.05 using a twotailed test. A Pearson correlation coefficient (r) of 0.9918 was obtained with a significance of (****), p-value < 0.0001. MMP−9 (ng/mL) Figure 2. Concentration of MMP-9 in the collection of tear samples from healthy controls, without any ocular disorder diagnosed, using ELISA (blue) as the gold standard technique and AbMAs (orange). MMP-9 concentration is represented as ng/mL for each individual. A gray line is plotted at 30 ng/mL of MMP-9 enzyme in tear, the threshold value at which higher concentrations are considered a sign of ocular inflammation. 2.3. Analysis of Pathological Samples Enzyme biomarker MMP-9 was also evaluated in the 20 patient samples using the AbMA developed technology. Concentration values of MMP-9 were obtained for each tear sample. P 1, P 3, P 4, P 6, P 7, P 8, P 9, P 10, P 11, P 13, P 14, and P 16 samples surpassed the pathological threshold established at 30 ng/mL of MMP-9 in the fluid. These 12 samples represent 60% of the tear collection from patients diagnosed with an ocular pathology used in this study (Figure 3). Figure 2. Concentration of MMP-9 in the collection of tear samples from healthy controls, without any ocular disorder diagnosed, using ELISA (blue) as the gold standard technique and AbMAs (orange). MMP-9 concentration is represented as ng/mL for each individual. A gray line is plotted at 30 ng/mL of MMP-9 enzyme in tear, the threshold value at which higher concentrations are considered a sign of ocular inflammation. 2.3. Analysis of Pathological Samples Enzyme biomarker MMP-9 was also evaluated in the 20 patient samples using the AbMA developed technology. Concentration values of MMP-9 were obtained for each tear sample. P 1, P 3, P 4, P 6, P 7, P 8, P 9, P 10, P 11, P 13, P 14, and P 16 samples surpassed the pathological threshold established at 30 ng/mL of MMP-9 in the fluid. These 12 samples represent 60% of the tear collection from patients diagnosed with an ocular pathology used in this study (Figure 3). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 18 MMP−9 (ng/mL) Figure 3. ng/mL of MMP-9 in ocular inflammation patient tear samples quantified using AbMA. A gray line is plotted at 30 ng/mL of MMP-9 enzyme in tear, the threshold value at which higher concentrations are considered a sign of ocular inflammation. In addition, the MMP-9 concentration in each tear sample was compared between healthy and pathological subgroups (Figure 4). The normality of the samples was evaluated using a Shapiro–Wilk test; setting the significance at α = 0.05 using a two-tailed test, the groups did not follow a Gaussian distribution. The patient group was divided into a glaucoma group, a cataract group, and the other pathologies group, englobing MGD, allergy, as well as DE. Cliff’s delta values were calculated for quantifying the amount of difference between control and pathological groups. The Cliff’s delta value when comparing the healthy group and the other pathologies group was δ = −0.208; for cataracts patients versus healthy individuals, it was δ = 0.438; and for glaucoma patients versus healthy individuals, it was δ = 0.583. Figure 3. ng/mL of MMP-9 in ocular inflammation patient tear samples quantified using AbMA. A gray line is plotted at 30 ng/mL of MMP-9 enzyme in tear, the threshold value at which higher concentrations are considered a sign of ocular inflammation. Int. J. Mol. Sci. 2022,23, 5639 7 of 16 In addition, the MMP-9 concentration in each tear sample was compared between healthy and pathological subgroups (Figure 4). The normality of the samples was evaluated using a Shapiro–Wilk test; setting the significance at α = 0.05 using a two-tailed test, the groups did not follow a Gaussian distribution. The patient group was divided into a glaucoma group, a cataract group, and the other pathologies group, englobing MGD, allergy, as well as DE. Cliff’s delta values were calculated for quantifying the amount of difference between control and pathological groups. The Cliff’s delta value when comparing the healthy group and the other pathologies group was δ = − 0.208; for cataracts patients versus healthy individuals, it was δ = 0.438; and for glaucoma patients versus healthy individuals, it was δ= 0.583. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 18 MMP−9 (ng/mL) MMP−9 (ng/mL) MMP−9 (ng/mL) Figure 4. Tear MMP-9 concentration differences in the groups of patients suffering ocular inflammation versus the group of healthy controls. Cliff’s delta values are displayed for each comparison. (A) Differences between healthy controls and MGD, DE, and allergy patients (other pathologies group). (B) Differences between healthy controls and cataracts patients. (C) Differences between healthy controls and glaucoma patients. Figure 4. Tear MMP-9 concentration differences in the groups of patients suffering ocular inflammation versus the group of healthy controls. Cliff’s delta values are displayed for each comparison. ( A ) Differences between healthy controls and MGD, DE, and allergy patients (other pathologies group). ( B ) Differences between healthy controls and cataracts patients. ( C ) Differences between healthy controls and glaucoma patients. Int. J. Mol. Sci. 2022,23, 5639 8 of 16 No differences were observed when comparing tear MMP-9 concentrations between age, gender, and Schirmer’s test results (data not shown). When analyzing the inflammation biomarker in the samples based on the established pathological threshold (30 ng/mL), differences were observed between the groups (Figure 5). Both healthy controls and other pathologies groups presented MMP-9 concentrations mainly below the threshold; contrarily, the cataracts and glaucoma groups presented tear MMP-9 values mostly over 30 ng/mL. In summary, 88% of the healthy controls and 83% of the other pathologies group samples were under the threshold; 75% of cataracts and 83% of glaucoma tear samples were over the threshold. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 18 No differences were observed when comparing tear MMP-9 concentrations between age, gender, and Schirmer’s test results (data not shown). When analyzing the inflammation biomarker in the samples based on the established pathological threshold (30 ng/mL), differences were observed between the groups (Figure 5). Both healthy controls and other pathologies groups presented MMP-9 concentrations mainly below the threshold; contrarily, the cataracts and glaucoma groups presented tear MMP-9 values mostly over 30 ng/mL. In summary, 88% of the healthy controls and 83% of the other pathologies group samples were under the threshold; 75% of cataracts and 83% of glaucoma tear samples were over the threshold. (>30 ng/mL) 1165 (≤30 ng/mL) 7521 Figure 5. Number of individuals or patients over and below the pathological threshold in the different groups. 2.4. Evaluation of the Diagnostic Performance of the Test Finally, in order to evaluate the diagnostic ability of the developed AbMA test, a receiver operating characteristic (ROC) curve analysis was performed [42,43]. The optimal threshold value for the test was determined using this analysis, resulting in 33.6 ng/mL of tear MMP-9 using our data. In addition, a confusion matrix was set up (Figure 6) following the indications of the guide The Fitness for Purpose of Analytical Methods of Eurachem [44], assessing the sensitivity at 60%, the specificity at 88%, and the accuracy at 68%. Figure 5. Number of individuals or patients over and below the pathological threshold in the different groups. 2.4. Evaluation of the Diagnostic Performance of the Test Finally, in order to evaluate the diagnostic ability of the developed AbMA test, a receiver operating characteristic (ROC) curve analysis was performed [ 42 , 43 ]. The optimal threshold value for the test was determined using this analysis, resulting in 33.6 ng/mL of tear MMP-9 using our data. In addition, a confusion matrix was set up (Figure 6) following the indications of the guide The Fitness for Purpose of Analytical Methods of Eurachem [ 44 ], assessing the sensitivity at 60%, the specificity at 88%, and the accuracy at 68%. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 of 18 Figure 6. Confusion matrix of tear MMP-9 analysis over the different samples. True positive (TP), false positive (FP), false negative (FN), and true negative (TN) rates are detailed. Sensitivity is calculated as TP/(TP + FN), specificity as TN/(TN + FP), and accuracy as (TP + TN)/(TP + FP + FN + TN). 3. Discussion We developed an AbMA test immobilizing an anti-MMP-9 IgG antibody and establishing a detection protocol for the quantification of MMP-9 in tears. The purpose of this test is the detection and quantification of human MMP-9 in tear samples as an instrument for ocular inflammation evaluation. The data obtained demonstrated once again that MMP-9 is a good biomarker of inflammation in various ocular pathologies [45], as well as validating microarray immunodetection technology as a diagnostic tool in the detection of MMP-9 and the monitoring of patients with inflammation-related pathologies such as glaucoma. Our results validated the developed AbMA test for the detection and quantification of human MMP-9 in tear samples as an instrument for ocular inflammation evaluation. For this purpose, eight tear samples from healthy individuals were collected, as well as 20 tear samples from patients suffering various ocular inflammatory conditions: cataracts, glaucoma, meibomian gland dysfunction, allergy, and DE. All 28 tear samples were defined in terms of donor age, gender, condition and Schirmer’s test results. Firstly, MMP9 concentration was determined with the currently used gold standard in this area, the ELISA technique, in order to evaluate if there is a positive correlation between this technique and the AbMA test, since the AbMA test aims to be a new method for biomarker quantification. The concentration of MMP-9 in the tear collection from healthy donors was quantified by ELISA and AbMAs according to their specific protocols. A statistically significant correlation in the obtained values was observed, with a Pearson correlation coefficient of 0.9918 and a p-value < 0.0001. These results are in agreement with previous studies using this technology in human tear [25], pointing out that AbMAs are a useful and reliable tool for MMP-9 quantification in human tear samples. Once the test was validated, tear samples of 20 patients with ocular inflammation were studied. These patients were suffering from different diseases related to inflammation (cataracts, glaucoma, MGD, allergy, and DE) [46]. MMP-9 biomarker was quantified using the developed AbMAs, which resulted in 60% of the samples surpassing the pathological threshold. This was established at 30 ng/mL based on the literature [47]; higher concentrations of the enzyme biomarker in tear samples were associated with ocular surface inflammation [26]. The observed global elevation of MMP-9 in the pathological samples was reasonable due to the inflammatory characteristics of this biomarker, which increases in response to stress when cytokine or chemokine pathways Figure 6. Confusion matrix of tear MMP-9 analysis over the different samples. True positive (TP), false positive (FP), false negative (FN), and true negative (TN) rates are detailed. Sensitivity is calculated as TP/(TP + FN), specificity as TN/(TN + FP), and accuracy as (TP + TN)/(TP + FP + FN + TN). Int. J. Mol. Sci. 2022,23, 5639 9 of 16 3. Discussion We developed an AbMA test immobilizing an anti-MMP-9 IgG antibody and establishing a detection protocol for the quantification of MMP-9 in tears. The purpose of this test is the detection and quantification of human MMP-9 in tear samples as an instrument for ocular inflammation evaluation. The data obtained demonstrated once again that MMP-9 is a good biomarker of inflammation in various ocular pathologies [ 45 ], as well as validating microarray immunodetection technology as a diagnostic tool in the detection of MMP-9 and the monitoring of patients with inflammation-related pathologies such as glaucoma. Our results validated the developed AbMA test for the detection and quantification of human MMP-9 in tear samples as an instrument for ocular inflammation evaluation. For this purpose, eight tear samples from healthy individuals were collected, as well as 20 tear samples from patients suffering various ocular inflammatory conditions: cataracts, glaucoma, meibomian gland dysfunction, allergy, and DE. All 28 tear samples were defined in terms of donor age, gender, condition and Schirmer’s test results. Firstly, MMP-9 concentration was determined with the currently used gold standard in this area, the ELISA technique, in order to evaluate if there is a positive correlation between this technique and the AbMA test, since the AbMA test aims to be a new method for biomarker quantification. The concentration of MMP-9 in the tear collection from healthy donors was quantified by ELISA and AbMAs according to their specific protocols. A statistically significant correlation in the obtained values was observed, with a Pearson correlation coefficient of 0.9918 and a p-value < 0.0001. These results are in agreement with previous studies using this technology in human tear [ 25 ], pointing out that AbMAs are a useful and reliable tool for MMP-9 quantification in human tear samples. Once the test was validated, tear samples of 20 patients with ocular inflammation were studied. These patients were suffering from different diseases related to inflammation (cataracts, glaucoma, MGD, allergy, and DE) [ 46 ]. MMP-9 biomarker was quantified using the developed AbMAs, which resulted in 60% of the samples surpassing the pathological threshold. This was established at 30 ng/mL based on the literature [ 47 ]; higher concentrations of the enzyme biomarker in tear samples were associated with ocular surface inflammation [ 26 ]. The observed global elevation of MMP-9 in the pathological samples was reasonable due to the inflammatory characteristics of this biomarker, which increases in response to stress when cytokine or chemokine pathways are activated. When MMP-9 values were analyzed for each subject within the four study groups, the MMP-9 threshold was exceeded by only 12% and 17% of individuals in the control and other pathology groups, respectively. However, 75% and 83% of patients in the glaucoma and cataract groups, respectively, had a tear MMP-9 concentration above the pathological threshold of 30 ng/mL, denoting ocular surface inflammation. Similar results were described by Kim and coworkers when they reported that approximately 72% of glaucoma patients displayed a high concentration of tear MMP-9 (over 40 ng/mL); however, when studying a control group of 47 healthy subjects, only about 32% of them showed an increase in this biomarker [ 48 ]. Again, this validated the AbMA results in concordance with the literature [ 25 ], highlighting the importance of MMP-9 as a tear biomarker of ocular surface inflammation. In order to assess the diagnosis capability of the test when studying each pathology, tear MMP-9 concentration was compared among groups and Cliff’s value was calculated as a useful complementary analysis for the corresponding hypothesis testing [ 48 ]. When compared with the healthy controls, the other pathologies, cataracts, and glaucoma groups obtained δ values of − 0.208, 0.438, and 0.583, respectively. Values over a δ = 0.474 meant a large difference between the two groups [ 48 ]. Taking this into account, the glaucoma group was the one with the highest δ when compared with the controls, which indicated a major difference in the presence of this biomarker, preliminarily pointing out that the developed technology was able to detect ocular inflammation pathology-related states. Likewise, the cataracts group presented a δ = 0.438, meaning a medium (defined Int. J. Mol. Sci. 2022,23, 5639 16 of 16 58. 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