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목적 : The primary goal of present study is to validate the tear matrix metalloproteinase 9 (MMP-9) in-situ immunoassay for detection of tear MMP-9 and to elucidate the characteristics of assay that could affect the results or its interpretation in vitro.
방법 : This experiment was carried out using a commercially available tear MMP-9 in-situ immunoassay (InflammmaDry) Assay reproducibility and sensitivity were analyzed. To elucidate the effect of sample volume on sensitivity, 2.5, 5, 10, and 20 μl of rhMMP-9 at concentrations of 0, 12.5, 25, 50, 100, 200, 500, and 1000 ng/ml, respectively. To determine the interaction of commercial eye drops with immunoassay, 10 μl of 200 ng/ml rhMMP-9 and 10 μl of each eye drop were consecutively applied to the sampling fleece. The test line of result window was photographed and analyzed by Image J.
결과 : After applying 10 μl of sample, despite positive result of test line could be identified on Image J measurement at 50 ng/ml concentration, but test line was only observable at 100 ng/ml concentration. By 20 μl of sample volume, a detection band could be identified at a concentration of 25 ng/ml on image densitometry and was observable visually at 50 ng/ml of rhMMP-9. With the 10 μl sample volume, densitometry results of the band showed linear dose-response pattern. By the 20 μl sample, band densitometry showed linear pattern increment until 500 ng/ml, but fully saturated after 500 ng/ml.
결론 : To perform proper detection of MMP-9 over 40 ng/ml concentration by MMP-9 in-situ immunoassay, sample volume is most crucial factor. MMP-9 in-situ immunoassay showed high reliability with 20 μl sample volume for detecting more than 40 ng/ml of rhMMP-9.
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