Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Summary

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

Abstract

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Introduction

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that is present in neuronal fibers with perivascular localization as well as non-neuronal tissues. The two forms of CGRP, α- and β-CGRP, share more than 90% homology and share physiologic functions; however, αCGRP is found in the central and peripheral nervous system, while βCGRP is found in the enteric nervous system^1,2. Upon nociceptor activation and calcium-dependent exocytosis, CGRP is released from neurons, inducing sterile neurogenic inflammation involving arterial vasodilatation and plasma protein extravasation^3,4,5,6,7. From here, CGRP appears in the postcapillary vessels and may be a biomarker for diseases that cause afferent nociceptive activation, such as migraine^8,9,10,11. Of note, CGRP has also been implicated in COVID-19 through its role in angiogenesis and immune modulation, and may predict unfavorable disease evolution^12,13. Thus, a protocol for the accurate quantification of CGRP in human plasma could have a broad value.

The most attention has perhaps been given to CGRP's role in migraine. Based on preclinical and clinical studies, CGRP has been put forth as a possible biomarker for migraine and as a target for treatment^{3,4,5,6,7,8,9,10}. Some studies have found an elevation of CGRP in cohorts with episodic migraine relative to control participants^10,14,15. The success of CGRP inhibitors in clinical trials for migraine headache treatment seem to implicate elevated CGRP as a causal factor for migraine headaches. However, not all investigators have corroborated these results^16,17,18,19. Moreover, the role of CGRP in non-headache symptoms of migraine has yet to be elucidated; the current work was motivated by a desire to understand the role of CGRP in vestibular symptoms of migraine.

Inconsistent CGRP immunoassay data in the literature could be due to several reasons. Firstly, the half-life of CGRP in the peripheral vasculature is 6.9 min²⁰, due to the activity of serine proteases²¹, insulin-degrading enzymes and other metalloproteases²², neutral endopeptidases²³, and endothelin-converting enzyme-1²⁴. Secondly, the variable technical details of the immunoassays used to quantify CGRP are not fully described in such studies. Finally, the lack of standardization of the immunoassay methodology complicates the picture even more.

This article describes a modified enzyme-linked immunosorbent assay (ELISA) protocol that allows for the purification and accurate quantification of α- and βCGRP in human plasma. The kit's antibodies are not cross-reactive with amylin, calcitonin, or substance P. This protocol has undergone the necessary validation experiments, such as spike and recovery and linearity of dilution, the data for which are presented here. Such a CGRP ELISA protocol that has undergone validation has not previously been fully described in the literature. This protocol can be used to quantify CGRP in human plasma in the context of migraine as well as cardiologic^2,25, dermatologic²⁶, obstetrical²⁷, rheumatologic^28,29, musculoskeletal^30,31, endocrine^32,33, and viral diseases^12,13 in which CGRP has been implicated.

Protocol

This protocol was developed using human plasma samples from consented individuals with approval from the Johns Hopkins Institutional Review Board (NA_00092491).

1. Sample collection and preparation

1. Collect 5 mL of whole blood from the antecubital vein via standard venipuncture methods³⁴ into a 6 mL vacutainer ethylenediaminetetraacetic acid (EDTA) collection tube.
2. Add 0.5 mL of aprotinin (10,000 KIU/mL) competitive serine protease inhibitor to the tube after collection to block the lysis of the target peptide. Invert the tube 10 times to allow the aprotinin to adequately mix with the blood. Afterward, store the tubes on ice until the next step.
3. Centrifuge the tube at 604 x g for 4 min at 4 °C within 60 min of blood collection.
4. Take off the plasma fraction with a pipette and transfer to 2 mL round-bottom sterile cryovials.
5. Immediately store the cryovials at -80 °C for up to 2 weeks.

2. Extraction of the plasma samples

1. Place an extraction cartridge inside a 15 mL conical centrifuge tube with the outer ridges of the cartridge supported by the outer ridges of the tube.
2. Activate the cartridge by first passing 5 mL of 100% methanol and then 10 mL of ultrapure water through the cartridge.
   1. As fluid is passed through the cartridge via gravity-driven flow, it will collect in the tube.
   2. Throw out excess fluid as it accumulates to ensure that the fluid does not engulf the cartridge.
   3. Ensure that the cartridge does not dry during the course of the experiment.
3. Dilute 250 µL of plasma with 750 µL of 4% acetic acid.
4. Pass the 1 mL of plasma and acetic acid solution slowly through the cartridge.
   NOTE: This step should take approximately 30 s via gravity-driven flow for each milliliter passed.
5. Wash the cartridge with 10 mL of 4% acetic acid. As done previously, throw out the excess fluid as it accumulates to ensure that the fluid does not engulf the cartridge.
6. After the last drop of acetic acid is released from the cartridge, remove the cartridge from the tube and place it in a new 15 mL conical centrifuge tube.
7. Prepare a 10:1 solution of 100% methanol and 4% acetic acid by mixing 2.7 mL of 4% acetic acid and 0.3 mL of 100% methanol. Use this to elute the CGRP by passing this solution through the cartridge 1 mL at a time. Pause for 25 min between each milliliter of solution passed. Do NOT throw out the eluent.
8. The tube will contain 3 mL of the eluted fluid 25 min after the last milliliter of the methanol and acetic acid solution has passed. Place each 1 mL of the eluent into a 1.7 mL microcentrifuge tube.
   NOTE: This should yield three microcentrifuge tubes from each cartridge.
9. Place each tube in a mini centrifuge for 1 s to ensure that all the eluent is at the bottom of each tube.
10. Dry all the samples by vacuum centrifugation at 4 °C (set to concentrator function, for aqueous solutions, at 1,400 rpm; the g-force varies depending on the position of the tubes and therefore ranges from 130-250 x g).
    NOTE: The time taken for the vacuum centrifuge to dry the samples will depend on the type of microcentrifuge tube used.
11. Immediately before proceeding to the assay procedure (step 4.1), reconstitute the previously dried sample with enzyme immunoassay (EIA) buffer (see Table of Materials) thawed to room temperature (RT) or 20 °C.
    ​NOTE: The volume of EIA buffer used to reconstitute the dried sample should be equal to the original sample volume, or 250 µL.

3. Preparation of the ELISA plate - blocking to limit non-specific binding

1. Add 250 µL of tris-buffered saline (TBS)/fish gelatin blocking buffer to each well on the plate.
2. Place a cover sheet over the plate and incubate for 2 h at RT.
3. Remove the cover sheet and empty the plate by inversion or aspiration using a multichannel pipette. Then, blot the plate on a paper towel to discard any trace of liquid.
4. Dry the plate by leaving the plate in a laminar flow hood for 10 min.
5. After the plate is visibly dry, place it in a grip-sealed foil pouch with a silica gel desiccant sachet and store the pouch at -20 °C for 24 h.
   ​NOTE: The plates should be used within 4 weeks of plate incubation.

4. Prior to the assay procedure

1. Prepare reagents (EIA buffer, CGRP standard, CGRP quality control, CGRP tracer, wash buffer) as per the kit instructions. Prepare Ellman's reagent only after the 16-20 h incubation period ends.
2. Thaw all samples and reagents to RT before performing the rest of the protocol.

5. Assay procedure

1. Rinse each well in the blocked plate five times with a kit-provided wash buffer (300 µL/well). For each rinse, pause 30 s after placing the wash buffer in the wells, then throw out the liquid or aspirate using a multichannel pipette.
2. After the final rinse, remove all the buffer from the wells by inversion (inverting the plate to expel the liquid within wells) or aspiration using a multichannel pipette. Blot the last drops onto a paper towel and tap the inverted plate until all the liquid is visibly removed from the wells.
3. Set aside at least two wells with no reagents or buffer to be known as "Blank" wells. Then, set aside at least two other wells for non-specific binding (NSB).
4. Dispense 100 µL of EIA buffer into each NSB well.
5. Dispense 100 µL of the CGRP standards in duplicate into appropriate wells.
6. Dispense 100 µL of samples (reconstituted in EIA buffer) and quality control in duplicate into appropriate wells.
7. Dispense 100 µL of CGRP tracer (anti-CGRP antibody attached to acetylcholinesterase) to each well containing NSB, CGRP standards, samples, and quality control. Do not dispense tracer to the "Blank" wells.
8. Cover the plate using a clear cover sheet, sealing each well such that the samples and reagents in each well do not evaporate significantly. Incubate the plate for 16-20 h at 4 °C.
9. After incubation is completed, reconstitute the Ellman's reagent as per the kit instructions.
10. Invert the plate to remove all liquid. Rinse each well (as described above) three times with 300 µL of wash buffer.
11. After the third rinse, remove the liquid from the plates, place the plate on a shaker plate, and shake at 120 rpm for 2 min.
12. Wash the plate an additional three times. After the final rinse, remove all buffer from the wells by inversion or aspiration using a multichannel pipette. Blot the last drops of liquid onto a paper towel and tap the inverted plate until all the liquid is visibly removed from the wells.
    NOTE: The additional three washes described in this step may not be necessary if using an ELISA microplate washer.
13. Dispense 200 µL of Ellman's reagent into each well (not including the "Blank" wells).
14. Cover the plate with a new cover sheet and wrap it in aluminum foil to prevent any light exposure. Incubate in the dark at RT for 1 h.
15. Ensure that there is no liquid on the backside of the plate that may confound the spectrophotometer reading by wiping the backside with a dry paper towel.
16. Read the plate for absorbance at 405 nm (yellow color).

6. Data analysis

1. Calculate the average absorbance for each "Blank", NSB, standard, quality control, and the samples.
2. Subtract the NSB average absorbance values from the standard, quality control, and sample absorbance values.
3. Plot absorbance on the y-axis and concentration on the x-axis. Construct a standard curve using a four-parameter logistic regression model.
4. Once the curve is constructed, use the curve's equation to determine the interpolated concentrations for quality control and samples, which can be read on the x-axis.
   NOTE: The standard curve is validated only if the calculated interpolated concentration for the quality control is within 25% of the expected concentration (usually 125 pg/mL, but see the label of the quality control vial).

Results

There are several key steps in the protocol that should be highlighted. Firstly, aprotinin, a serine protease inhibitor, must be added to whole blood samples immediately upon collection to prevent further enzymatic degradation of CGRP. Serine proteases have been shown to play a role in CGRP metabolism, and a previous study has also used aprotinin in quantifying CGRP in humans^21,35. If protease inhibitors are not used, and sample preparation takes longer than 60 m...

Discussion

This article describes a validated protocol allowing for the detection and quantification of CGRP in human plasma. This protocol was synthesized after commercial CGRP ELISA kits were found to not accurately quantify this molecule. After establishing a sample preparation protocol and a valid standard curve, spike and recovery and linearity of dilution experiments showed that the percentage of recoveries were much lower than expected. Similar results were found using a different commercial CGRP ELISA kit (Table 4

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.7 mL Safeseal microcentrifuge tube	Sorenson Bioscience, Inc.	11510
99% methanol	ThermoFisher Scientific	L13255.0F
15 mL conical centrifuge tube	Falcon	14-959-49B
2 mL round bottom sterile cryovials	CRYO.S	122263
4% acetic acid	ThermoFisher Scientific	035572.K2
6.0 mL Vacutainer EDTA collection tube	BD	367863
Allegra 64R benchtop centrifuge	Beckman Coulter, Inc.	367586
Aprotinin	VWR	76344-814
CGRP (human) ELISA kit	Bertin Bioreagent	A05481
CGRP stock	Bertin Bioreagent
EIA Buffer	Bertin Bioreagent	A07000
Ellman's Reagent	Bertin Bioreagent	A09000_49+1
Multichannel pipettes	ThermoFisher Scientific	4661180N
Oasis HLB 3 cc Vac Cartridges	Waters	WAT094226
Orbital Shaker	Bellco	7744-01010
Precision micropipettes	ThermoFisher Scientific	F144055MG
SpectraMax M Series Multi-Mode Microplate reader	Molecular Devices	Part Number M2
TBS/Fish Gelatin	Bioworld, from Fischer Scientific	50-199-167
Ultrapure water ELISA Grade	Bertin Bioreagent	A07001
Vacufuge plus - Centrifuge Concentrator	Eppendorf	22820109
Wash Buffer	Bertin Bioreagent	A17000