Studies on the Optimization of Neuropeptides Detection in the Human Tear Film

Abstract

Introduction: Dry Eye Disease (DED) stems from a disruption of the homeostasis of the tear film (TF), a thin layer of fluid covering the ocular surface. The TF consists of numerous constituents that include proteins, lipids, mucins, water, electrolytes, immunoglobulins, vitamins, cytokines, and neuropeptides. An imbalance in any of these constituents could result in an unstable tear film, contributing to the pathophysiology of DED. Among these factors, neuropeptides, small proteinaceous substances produced and released by neurons through regulated secretory routes, may have a role in the pathophysiology of DED. To understand the impact of the disease on the concentrations of calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) in the tear film, it is important to first understand the sample collection and quantification methods. The purpose of this thesis was to optimize a method to quantify the concentration of four neuropeptides using enzyme linked immunosorbent assay (ELISA). a common laboratory technique used to quantify the concentration of neuropeptides in the tear film. ELISAs have been used to determine the quantity of different components in blood and other bodily fluids in the human tear film. The aims of each chapter were as follows: Chapter 3: To determine the variability of two tear collection methods, basal tear collection and flush tear collection, for quantifying SP, CGRP, VIP, and NPY, and to quantify the day-to-day variability of these neuropeptides. Chapter 4: To assess the validity of a commercially available ELISA kits for the quantification of neuropeptides. Chapter 5: To examine the measurement variability of two commercially available ELISA kits for the quantification of SP.

Methods: Chapter 3: Basal and flush tears (following instillation of 20 μL of saline on the ocular surface) of 8 healthy participants were collected from the right and left eyes respectively, using glass microcapillary tubes on two consecutive days. The concentrations of the four neuropeptides in the tears were determined using ELISA, for both collection methods, and for both days. Chapter 4: Basal tears (5 µL) were collected from the temporal canthus of each eye of 3 healthy participants using glass microcapillary tubes. To assess the validity of the ELISA kit used in Chapter 3, two experiments were performed: a spike and recovery experiment, followed by a serial dilution response. In the spike and recovery experiment, 2 μL of tears from each participant were diluted in 108 μL of three known concentrations of SP, CGRP, and NPY (1 pg/mL, 10 pg/mL, and 100 pg/mL). The concentrations of neuropeptides were quantified using ELISA and the percent recovery was calculated. In the serial dilution response experiment, 4 μL of tears were collected from a single participant and was spiked into a known concentration of NPY (100 pg/mL). Serial dilutions (1:2, 1:4 and 1:8) were conducted and the percent recovery was calculated. Multiple troubleshooting and optimizing experiments to conducted to examine the effect of using a blocking agent, C18 pipette tip column, protease inhibitor, and the effect of freeze storage on neuropeptide quantification. Chapter 5: SP from Phoenix Pharmaceuticals, SP from Cayman Chemicals and SP from Sigma-Aldrich were each formulated at 0.5 mg/mL. Their UV absorbance profile from 200 nm to 300 nm was obtained using SoftMax Pro 5.4.1 software on a SPECTRAmax M5e ROM v2.1.35. The two SP from Phoenix Pharmaceuticals and SP from Sigma-Aldrich were formulated at various concentrations (500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL, 15.6 pg/mL, to 7.8 pg/mL and 3.9 pg/mL) and were quantified using two different ELISA kits (Phoenix Pharmaceuticals, Cayman Chemicals). A Bland Altman plot was used to quantify the agreement of the two SP, quantified by each of the ELISA kits.

Results: Chapter 3: There was no significant difference in the concentrations of CGRP, SP, NPY, and VIP between the two collection methods (all P > 0.19). The difference in concentrations of CGRP, SP, NPY, and VIP between the two study days was also not significant (P > 0.06 for all tests). Chapter 4: The percent recovery for spike and recovery experiment ranged between 63,953.15% to 13.74% for CGRP, 676.17% to 5.21% for SP and 412.42% to 7.51% for NPY. The initial concentration of NPY was 208 pg/mL and after the 1:2, 1:4, and 1:8 dilution, the observed recovery was 188.40 pg/mL for the 1:2 dilution, 153.60 pg/mL for the 1:4 dilution, and 204.28 pg/mL for the 1:8 dilution. In the troubleshooting experiments; there were minimal differences in the concentration of SP associated with the use of a blocking agent; there was a reduction in VIP when processed using C18 column pipette tips; using protease inhibitors reduced the amount of VIP recovered; the amount of VIP recovered was reduced in the presence of albumin; a higher amount of SP was recovered in freshly collected tears compared to tears which were stored frozen for four months. Chapter 5: A similar absorbance profile was observed for SP from Phoenix Pharmaceuticals and SP from sigma Aldrich. A trend toward higher variability was observed at lower concentrations of SP. The Bland Altman plot shows a mean difference of -3.36%, and a 95% limits of agreement of [-10.75, 4.01] for the Phoenix Pharmaceuticals kit, and a mean difference was -9.70%, and the 95% limits of agreement were [-14.61, -4.79] for the Cayman Chemicals kit.

Conclusion:

Chapter 3: It was anticipated that diluting to facilitate collection using the flush tears method would have yielded a lower concentration than the basal tears collection method. However, the ELISA kits found no significant difference between the two collection methods. Chapter 4: High variation was observed in the recovered values of both the spike and recovery and the serial dilution response experiments. The troubleshooting experiments have provided some optimization steps to consider for tear sample collection and processing for the detection for neuropeptides. Chapter 5: The agreements of two SP standards quantified with two different SP ELISA kits were poor. Greater variability in fluorescence and absorbance units was associated with lower concentrations of SP, highlighting the difficulty in quantifying SP near the detection limit of the kits.