Abstract
We aimed to validate a tear MMP-9 in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation. Three factors were examined: sample concentration, volume, and time. Recombinant human (rh) MMP-9 (10 or 20 μl; 0, 12.5, 25, 50, 100, 200, 500, and 1,000 ng/ml) was applied to the kit and the detection limit and assay reproducibility were examined. At a rhMMP-9 volume of 10 μl (≥ 50 ng/ml), all positive results were identified by densitometry at 10 and 20 min; however, after 20 min, more than half of the nine ophthalmologists interpreted a positive result. At a rhMMP-9 volume of 20 μl (≥ 25 ng/ml), ophthalmologists and densitometry identified almost all test lines at 10 and 20 min. At 10 μl, densitometry showed a linear dose–response pattern. At 20 μl, densitometry showed a linear dose–response pattern at concentrations up to 500 ng/ml; however, full saturation was achieved at concentrations ≥ 500 ng/ml. When the same amount of rhMMP-9 was applied, the density result increased significantly upon doubling of the solvent volume (i.e., by adding the same volume of PBS to a sample). InflammaDry showed a high inter- and intra-assay coefficient of variation at 10 min (28.4% and 24.7%, respectively). The results of the MMP-9 in-situ immunoassay varied significantly depending on sample volume. Therefore, when interpreting the results, careful attention must be paid to tear volume.
Highlights
We aimed to validate a tear matrix metalloproteinase 9 (MMP-9) in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation
When a 10 μl sample volume was tested, more than half of the nine ophthalmologists were able to identify a positive band at a concentration of 100 ng/ml after 10 min and at a concentration of 50 ng/ml after 20 min (Fig. 2A)
When a 20 μl sample volume was tested, more than half of the nine ophthalmologists identified a positive band at 25 ng/ml after 10 min, and all identified a positive band after 20 min (Fig. 2B)
Summary
We aimed to validate a tear MMP-9 in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation. First-line diagnostic tools for dry eye include tear break-up time, the Schirmer test, symptom scoring questionnaires, and slit lamp biomicroscopy using fluorescein or lissamine green. Two diagnostic modalities were introduced for objective evaluation of tear hyperosmolarity and matrix metalloproteinase 9 (MMP-9) levels (a measure of ocular surface inflammation)[5,6,7] In the latter case, InflammaDry (Quidel Corporation, San Diego, CA, USA) is a disposable single-use immunoassay designed to detect active and latent forms human MMP-9 in tears[8]. Clinical interpretation of the faint test (red) line during examination can be ambiguous, because conditions involving deficiency of aqueous tears (such as Sjögren syndrome[19] or ocular graft-versus-host disease20) yield negative results despite severe signs and symptoms of dry eye. The present study aimed to validate the MMP-9 immunoassay by examining the effects of time, sample concentration, sample volume, and drug interaction on the results
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