Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips

Hideki Miyake, Naoto Mori, Hidetoshi Mano, Takahiro Imanaka, Masatsugu Nakamura Research and Development Division, Santen Pharmaceutical Co., Ltd., Osaka, Japan Purpose: Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye d...

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Autores principales:	Miyake H, Mori N, Mano H, Imanaka T, Nakamura M
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2018
Materias:	mucin MUC5AC Schirmer test dry eye tears assay Ophthalmology RE1-994
Acceso en línea:	https://doaj.org/article/127b4a9b90e74abc9fb754716bac2b87
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spelling	oai:doaj.org-article:127b4a9b90e74abc9fb754716bac2b872021-12-02T07:28:37ZDevelopment of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips1177-5483https://doaj.org/article/127b4a9b90e74abc9fb754716bac2b872018-08-01T00:00:00Zhttps://www.dovepress.com/development-of-a-highly-sensitive-and-reliable-enzyme-linked-immunosor-peer-reviewed-article-OPTHhttps://doaj.org/toc/1177-5483Hideki Miyake, Naoto Mori, Hidetoshi Mano, Takahiro Imanaka, Masatsugu Nakamura Research and Development Division, Santen Pharmaceutical Co., Ltd., Osaka, Japan Purpose: Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA. Methods: MUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined. Results: MUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%. Conclusion: We developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients. Keywords: mucin, MUC5AC, Schirmer test, dry eye, tears, assayMiyake HMori NMano HImanaka TNakamura MDove Medical PressarticlemucinMUC5ACSchirmer testdry eyetearsassayOphthalmologyRE1-994ENClinical Ophthalmology, Vol Volume 12, Pp 1571-1580 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	mucin MUC5AC Schirmer test dry eye tears assay Ophthalmology RE1-994
spellingShingle	mucin MUC5AC Schirmer test dry eye tears assay Ophthalmology RE1-994 Miyake H Mori N Mano H Imanaka T Nakamura M Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
description	Hideki Miyake, Naoto Mori, Hidetoshi Mano, Takahiro Imanaka, Masatsugu Nakamura Research and Development Division, Santen Pharmaceutical Co., Ltd., Osaka, Japan Purpose: Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA. Methods: MUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined. Results: MUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%. Conclusion: We developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients. Keywords: mucin, MUC5AC, Schirmer test, dry eye, tears, assay
format	article
author	Miyake H Mori N Mano H Imanaka T Nakamura M
author_facet	Miyake H Mori N Mano H Imanaka T Nakamura M
author_sort	Miyake H
title	Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
title_short	Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
title_full	Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
title_fullStr	Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
title_full_unstemmed	Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips
title_sort	development of a highly sensitive and reliable enzyme-linked immunosorbent assay for muc5ac in human tears extracted from schirmer strips
publisher	Dove Medical Press
publishDate	2018
url	https://doaj.org/article/127b4a9b90e74abc9fb754716bac2b87
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Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips

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