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

Our research focuses on women's health in ocular surface diseases, an adaptation of ELISA for sex hormone quantification in basal tear fluid. Traditional ELISAs require large sample volumes, making tear fluid analysis challenging. This protocol enables hormone or protein quantification from limited tear samples.

Begin by obtaining appropriate microcapillary tubes to collect tears and a 1.5 milliliter microcentrifuge tube for tear film storage. To collect the basal tears, instruct the participant to look straight ahead or slightly upward. Then gently place the microcapillary tube opening in the tear meniscus near the lateral canthus, avoiding lid and conjunctival contact to minimize reflex tearing.

Fill the tube with tears until it's full. Occlude the hole on the rubber squeeze bulb. Insert the microcapillary tube into the bulb and place the open end into the microcentrifuge tube.

Gently squeeze the bulb to release the tear into the microcentrifuge tube. Close the microcentrifuge tube cap to prevent evaporation. Spin the tube at 2000 G for three to five seconds to collect the sample at the bottom.

Then store the sample at 80 degrees Celsius for preserving analyte stability prior to analysis. Obtain all required reagents and materials and let them reach room temperature. Aliquot only the required amounts of ready to use standards and controls to minimize contamination and repeated temperature changes.

Prepare six standard solutions labeled as number zero to five. Include one control sample provided with the kit. Keep the frozen samples on ice for thawing.

Spin down the tier samples to ensure samples are at the bottom of the tube. Mix all standards, controls, and samples prior to pipetting into the wells for homogeneous distribution. Add 20 microliters of each standard.

Control and sample into wells. Add 160 microliters of 17 beta estradiol HRP conjugate to the testing wells. Then cover the plate with aluminum foil, or use the provided cover foil from the kit, and incubate it at 37 degrees Celsius for two hours.

In the meantime, prepare wash buffer from 10X washing solution. After incubation, remove the aluminum foil from the plate and aspirate the liquid from the wells. Wash each well three times with the wash buffer for at least five seconds each.

Add 100 microliters of TMB substrate solution into all wells. Cover the whole plate with aluminum foil and incubate at room temperature for 30 minutes. Post-incubation, add 100 microliters of stop solution into the wells, and observe the reagent color change from blue to yellow.

Then shake the microplate gently. Measure the absorbence at 450 nanometers within 30 minutes of adding the stop solution. Then calculate the concentration of 17 beta estradiol using appropriate tools.

In the present study, 20 women aged between 43 and 66 years with and without dry eye disease or DED were included. The average tear film 17 beta estradiol concentrations in older women without DED were approximately 66.59 picograms per milliliter and 47.07 picograms per milliliter in older women with DED. The percent coefficient of variation for estradiol measurements was 1.0%for non-DED samples and 6.65%for DED samples confirming assay reproducibility.

This protocol allows researchers to measure sex hormone concentrations using ELISA and basal tear fluid collected by using microcapillary tubes. The tear film provides a non-invasive, biologically rich source of biomarkers for ocular surface research. However, the major challenge with tear analysis is the limited tear volume that can be collected, which often necessitates sample collection across multiple visits, which inherently introduces biological variability that needs to be kept in mind when doing this type of work.

Future studies can use this protocol to investigate how tear hormone levels correlate with diseases or even monitor treatment effectiveness.