Fig 1: Concentration-band density relationship, as evaluated by densitometry. (A) Using 10 µl of rhMMP-9, a positive test line was detectable at concentrations = 50 ng/ml. (B) Using 20 µl of rhMMP-9, a positive band was detectable at concentrations = 25 ng/ml. (C) Using 10 µl of rhMMP-9, a linear dose–response relationship was evident. (D) Using 20 µl of rhMMP-9, a linear dose–response relationship was observed up to 500 ng/ml; however, the results reached a plateau above 500 ng/ml. (E,F) Visual appearance of the MMP-9 immunoassay at 10 and 20 min; visibility of the control and test lines at different concentrations of rhMMP-9 in 10 (E) or 20 (F) µl of sample. Data are expressed as the mean ± SD.
Fig 2: Additional experiments demonstrating the volume-dependency of the MMP-9 immunoassay. (A) Visual appearance of the MMP-9 immunoassay before and after addition of diluent (phosphate buffered saline, PBS). (B) Application of additional diluent after initial application of 10 µl of sample (50 or 100 ng/ml of rhMMP-9) to the sample fleece. Additional diluent (10 µl) increased the band density of 50 or 100 ng/ml of rhMMP-9 (n = 3). Data are expressed as the mean ± SD and evaluated using a paired t-test.
Fig 3: Percentage of ophthalmologists who identified a band positively at different concentrations. Nine ophthalmologists interpreted the MMP-9 immunoassay results. Each concentration comprised triplicate samples (total assays, n = 27). (A) At a sample volume of 10 µl, more than half of the ophthalmologists were able to detect a positive band at 50 ng/ml after 20 min. (B) At a sample volume of 20 µl, nearly all ophthalmologists detected a positive band at 25 ng/ml after 10 and 20 min.
Fig 4: Standard calibration curve showing the concentration-band density relationship using various concentrations of recombinant human (rh) pro-matrix metalloproteinase (MMP)-9 solutions. (A) There are slow progression sections below 100 ng/mL and over 750 ng/mL of concentration while there is a slow progression section between concentrations of 100 ng/mL and 750 ng/mL. The values in each concentration is an average value from 3 independent samples of the same concentration. (B) Representative photographs of readout windows of the point-of-care matrix metalloproteinase (MMP)-9 immunoassay at the different concentrations of rh MMP-9 from 0 ng/mL to 5000 ng/mL.
Fig 5: Effects of different eye drops on band density in the MMP-9 immunoassay. The density of the test line was evaluated after application of 10 µl (200 ng/ml) rhMMP-9, followed by 10 µl of each eye drop sample. The two dotted lines represent the average density of the control band (20 µl, 200 ng/ml rhMMP-9) after 10 and 20 min, respectively.
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