This modified ELISA protocol allows for the purification and accurate quantification of CGRP in human plasma. It has undergone necessary validation experiments and can serve to standardize future quantification methodologies. This protocol offers the advantages of including sample processing before significant protease degradation, purification via solid phase extraction, and limitation of non-specific binding by application of blocking buffer to the microtiter plates.
CGRP plays a punitive role in the pathophysiology of migraine and maybe a candidate for biomarker status. It has also been implicated in cardiologic, dermatologic, viral, and even COVID-19 related disease processes. To begin, collect 5 milliliters of whole blood from the antecubital vein through standard venipuncture methods into a 6 milliliter vacutainer EDTA tube.
To block the lysis of the target peptide, add 0.5 milliliters of aprotinin competitive serine protease inhibitor. Invert the tube 10 times and store it on ice until the next step. Then centrifuge the tube at 600 4g for four minutes at 4 degrees Celsius.
And transfer the plasma fraction to a 2 milliliter round bottom sterile cryo vial. Immediately store the cryo vial at minus 80 degrees Celsius for up to two weeks. For extracting the plasma sample, place an extraction cartridge inside a 15 milliliter conical centrifuge tube.
Pass 5 milliliters of 100%methanol, then 10 milliliters of ultrapure water through the cartridge to activate it. The fluid will be collected in the tube as it passes through the cartridge by gravity driven flow. Dispose of accumulated excess fluid to prevent it from engulfing the cartridge and ensure it does not dry out during the experiment.
Dilute 250 microliters of plasma in 750 microliters of 4%acetic acid, and slowly pass it through the cartridge. Then wash the cartridge with 10 milliliters of acetic acid. Once the excess fluid is removed, transfer the cartridge to a new 15 milliliter conical centrifuge tube.
Prepare 3 milliliters of methanol acetic acid solution to elute the calcitonin gene related peptide or CGRP. Pass 1 milliliter of it through the cartridge and wait for 25 minutes between each elution. After the last elution, the tube contains 3 milliliters of total eluant.
Transfer 1 milliliter of the eluant into a 1.7 milliliter micro centrifuge tube, and spin it in a mini centrifuge for one second to settle the eluant. Then dry the sample in a vacuum centrifuge at 1, 400 RPM and 4 degrees Celsius. Immediately before the assay, reconstitute the dried sample with thawed enzyme immunoassay or EIA buffer.
For plate blocking, add 250 microliters of TBS fish gelatin blocking buffer to each well. Place a cover sheet over the plate and incubate for two hours at room temperature. After incubation, remove the cover sheet and empty the plate by inverting it.
Then blot the plate on a paper towel to discard any trace liquid and dry it in a laminar flow hood for 10 minutes. Once the plate is visibly dry, place it in a grip sealed foil pouch with a silica gel desiccant sachet. Store it at minus 20 degrees Celsius for 24 hours.
Before performing the assay, thaw all samples and prepared reagents at room temperature. To rinse the wells of the block plate, add 300 microliters of wash buffer and let it sit for 30 seconds. Then discard the wash buffer and repeat rinsing five times.
After the last rinse, blot the plate as previously demonstrated. Next, dispense 100 microliters of EIA buffer to the dedicated two non-specific binding or NSB wells. Add 100 microliters of the CGRP standards, followed by the reconstituted sample and quality control in duplicate into appropriate wells.
Add 100 microliters of CGRP tracer to each well, except the blank wells. Finally, seal the plate using a transparent cover sheet, ensuring the samples and reagents do not evaporate, and incubate the plate for 16 to 20 hours at 4 degrees Celsius. When the incubation period is over, prepare Ellman's reagent as per the manufacturer's instructions.
Once the plate is decanted, wash each well three times as previously demonstrated. After the last rinse, shake the plate at 120 RPM for two minutes. Again, rinse the plate thrice and blot it dry.
Tap the inverted plate until the liquid is visibly removed from the wells. Then add 200 microliters of Ellman's reagent into each well except blank wells. Once the plate is covered, wrap it in aluminum foil to prevent any light exposure and incubate it in the dark at room temperature for one hour before recording the absorbance.
The present study demonstrated that the addition of the blocking step, the recovery rates in each CGRP spike sample were within the acceptable range. However, when the blocking step was excluded, the non-specific binding resulted in recovery rates exceeding the upper limit of 125%It is important to add aprotinin to hold blood samples shortly after collection to limit CGRP degradation. Additionally, the blocking buffer should be added to the microtiter plate prior to ELISA to further reduce non-specific binding.
So this protocol can be utilized to explore the candidacy of CGRP as a biomarker for vestibular migraine by quantifying levels of CGRP in the plasma patients with migraine and vestibular migraine versus controls.
