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

Abstract

PurposeReliable 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.MethodsMUC5AC 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.ResultsMUC5AC 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%.ConclusionWe 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.

Morphological features of the eyelid margin in relation to conjunctival goblet cells and MUC5AC in tear film and clinical tests of dry eye

“…The MUC5AC protein in the tear lm was quanti ed using an enzyme-linked immunoassay assay (ELISA) according to a previously reported method [14]. Tears were collected using unmarked Schirmer strips (Jin Ming, Tianjin, China) and frozen at − 80°C after the length of the strip wetting with tears was measured before the next step.…”

The Physiological Function and Potential Role of the Ubiquitous Na+/H+ Exchanger Isoform 8 (NHE8): An Overview Data

“…In the absence of NHE8, the tear volume is reduced by approximately 34%, along with increased focal inflammatory infiltration and foamy vacuoles within acinar cells in the lacrimal gland [7]. The conjunctival epithelial layer may also contribute to the reduction of tear volume since abnormal expression of small proline-rich protein 2H (Sprr2h) and transglutaminase 1 (Tgm1) were detected in the eyes of NHE8KO mice [97]. MUC5AC, primarily secreted mucin involved in the lubrication of the ocular surface, is produced by the goblet cells in the conjunctiva [97].…”

“…The conjunctival epithelial layer may also contribute to the reduction of tear volume since abnormal expression of small proline-rich protein 2H (Sprr2h) and transglutaminase 1 (Tgm1) were detected in the eyes of NHE8KO mice [97]. MUC5AC, primarily secreted mucin involved in the lubrication of the ocular surface, is produced by the goblet cells in the conjunctiva [97]. In the absence of NHE8, the expression of Muc5ac is reduced by 60%, suggesting a role of NHE8 in goblet cell function in the eye [97].…”

“…MUC5AC, primarily secreted mucin involved in the lubrication of the ocular surface, is produced by the goblet cells in the conjunctiva [97]. In the absence of NHE8, the expression of Muc5ac is reduced by 60%, suggesting a role of NHE8 in goblet cell function in the eye [97].…”

Contribution of Mucins towards the Physical Properties of the Tear Film: A Modern Update

“…Firstly, it was recently shown that the method of collection of tear samples (microcapillary, Schirmer strips, etc.) may result in very different amount of mucins, lipids, and other compounds in the specimen which considering the complex hetero-molecular interactions behind the non-Newtonian behavior of human tears may grossly impact the sample's performance and composition [24,25]. Secondly, the commercial mucin preparations used in the experiments of Tiffany (primarily bovine submaxillary mucin) have very different levels of N-and O-glycosylation compared to ocular mucins [6,7] which certainly will affect their hydrogen bonding pattern and rheological properties.…”