Sex Hormone Detection In Human Tear Film Using Enzyme-linked Immunosorbent Assays

The tear film is a complex, multi-layered fluid essential for maintaining ocular surface health, visual clarity, and protection against pathogens and environmental stressors. Composed primarily of lipids, aqueous fluid, soluble proteins, and mucins, it serves not only to lubricate and protect the eye but as a valuable diagnostic medium for ocular surface disorders such as dry eye disease (DED). Sex hormones, including androgens and estrogens, are known to influence tear film composition, but measurement of these hormones in tear fluid is limited by low sample volume and technical challenges. This protocol describes a step-by-step method for adapting commercially available enzyme-linked immunosorbent assay (ELISA) kits to detect and quantify 17β-estradiol and testosterone in basal tears collected using microcapillary tubes. The workflow incorporates optimized sample collection, dilution, and assay preparation strategies to enable analysis of low-volume tear samples. The protocol demonstrates the feasibility and reproducibility of hormone detection in tear fluid using adapted ELISA methods. Representative results are derived from pooled pilot samples and are intended to illustrate assay performance rather than biological differences. This approach may support future studies investigating tear fluid as a matrix for hormone measurement in ocular surface research.