This article outlines a method for quantifying immune mediators in nasal mucosal lining fluid using high-sensitivity electrochemiluminescence-based multiplexed arrays. The process involves sample preparation, including thawing, centrifugation, and the application of specific antibodies for detection.
Thaw the filter papers containing the absorbed nasal mucosal lining fluid on ice to prevent sample degradation.
The fluid comprises immune mediators, including chemokines and cytokines.
Immerse the filter papers in a buffer. Transfer the contents into tube filters. Centrifuge to elute the mucosal lining fluid from the filter paper.
Add the filtered nasal extract to the assay plate patterned with an array of mediator-specific capture antibodies.
The immune mediators bind to their corresponding antibodies.
Introduce detection antibodies labeled with a ruthenium complex, which bind to their respective mediators.
Add tris-based buffer containing tripropylamine, or TPA.
When a potential is applied across the electrodes, the ruthenium complex and TPA undergo oxidation.
The resulting TPA radical excites the ruthenium complex.
Upon returning to its ground state, the ruthenium complex emits photons.
The measured light intensity is proportional to the specific immune mediator concentration in the mucosal lining fluid.
For airway immune mediator quantification, place up to 10 samples from negative 80 degrees Celsius storage on ice, and record their identification numbers. When the samples have thawed, immerse both of the filter papers per subject in 150 microliters of freshly prepared buffer per filter paper.
Supplement it with one complete protease inhibitor tablet per 25 milliliters of buffer stock solution. Transfer the samples to a plate shaker at 400 RPM for 5 minutes. Transfer the buffer and filter papers into the cups of individual cellulose acetate tube filters.
Centrifuge the samples to collect the filtered nasal extract at the bottom of the microcentrifuge tubes. Then, remove the cups, and place the tubes on ice. Next, transfer 150-microliter aliquots of the nasal extract into individual wells of low-protein-binding storage plates, and store the plates at negative 80 degrees Celsius until analysis. Then, measure the concentration of the nasal immune mediators of interest by high-sensitivity electrochemiluminescence-based multiplexed array, according to standard assay protocols.
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