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Naoto Mori, Hideki Miyake, Hidetoshi Mano, Takahiro Imanaka, Masatsugu Nakamura, Takeshi Matsugi, Naveed K Shams; MUC5AC assay of extracts from Schirmer strips with human tear using a modified commercial ELISA kit. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4874.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment. Reliable measurement of human tear mucin 5AC (MUC5AC) is important for clarifying the pathophysiological role of MUC5AC in dry eye. Here, we developed an assay by optimizing MUC5AC extraction from Schirmer strips, improving the sensitivity and validating the use of a commercially available ELISA kit.
Healthy human tears were purchased from Lee Biosolutions, Inc., (Maryland Heights, MO). Informed consent was obtained before sample collection. Tears were spotted onto Schirmer strips and soaked in phosphate-buffered saline containing various concentrations of polysorbate 20 at room temperature and at 37°C. Extracts from strips were treated with neuraminidase to cleave sialic acids from MUC5AC. The ELISA kit for MUC5AC was purchased from Cloud-Clone Corp (Houston, TX). The ELISA plate was coated with blocking reagent (Block Ace Powder; DS Pharma Biomedical Co., Ltd. Osaka, Japan). This method was validated for extraction rate from Schirmer strips, limit of quantification, calibration range, and reproducibility.
MUC5AC extraction rate from Schirmer strips increased depending on polysorbate 20 concentration. Conversely, ELISA sensitivity decreased in proportion to polysorbate 20 concentration. The optimized concentration of polysorbate 20 was set to 0.05%. MUC5AC extraction rate from Schirmer strips was 89% with 0.05% polysorbate 20 at 37°C. Treatment with neuraminidase significantly increased the signal-to-noise ratio more than 8-fold in the ELISA compared with in the absence of the enzyme. Blocking before the incubation step decreased nonspecific adsorption of MUC5AC to the plate, showing good linearity of tear dilution. In the standard calibration, the lower limit of quantification for MUC5AC was 19.5 pg/mL.
We developed a reliable ELISA method for MUC5AC in extracts from Schirmer strips. This method is useful to detect MUC5AC in tears of patients with dry eye, and can be used to subcategorize mucin deficiency-type dry eye.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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