Fig 1: Representative images showing the processes for the quantification of the colorimetric density of the red band in the readout window of the point-of-care matrix metalloproteinase (MMP)-9 immunoassay. (A) First, the MMP-9-expressing red band was outlined with a black rectangle, then was cropped. The vertical length was sized into 200 pixels (Px). The image was converted to the 32-bit one and the area with relatively high color density was detected by auto-threshold. The red-colored area (asterisk) was estimated. (B) The representative 5 images in each grade before and after the color threshold.
Fig 2: Visual appearance of the MMP-9 immunoassay used to assess inter-assay (A) and intra-assay (B) precision at different sample concentrations and volumes (10 µl, 500 ng/ml rhMMP-9; 20 µl, 200 ng/ml rhMMP-9). Calculated values are shown (C). CV = coefficient of variation.
Fig 3: 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 4: 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 5: 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.
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