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

Pavan S. Krishnan; Fernando T. Zamuner; Carolyn M. Jenks; Johnny Y. Xie; Lisa Zhang; Mohammed Lehar; Neal S. Fedarko; Mariana Brait; John P. Carey

doi:10.3791/64775

JoVE Journal > Biochemistry

Biochemie

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

Published: June 16, 2023

doi:

10.3791/64775

Pavan S. Krishnan^1,2, Fernando T. Zamuner^1,3, Carolyn M. Jenks¹, Johnny Y. Xie⁴, Lisa Zhang⁵, Mohammed Lehar¹, Neal S. Fedarko⁶, Mariana Brait^1,3, John P. Carey¹

¹Department of Otolaryngology-Head & Neck Surgery,Johns Hopkins University School of Medicine, ²Virginia Commonwealth University School of Medicine, ³Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center,Johns Hopkins University School of Medicine, ⁴Department of Otolaryngology-Head and Neck Surgery,University of Michigan Medical School, ⁵Department of Otolaryngology-Head and Neck Surgery,The Ohio State University College of Medicine, ⁶ICTR Clinical Research Core Laboratory,Johns Hopkins University School of Medicine

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 system¹^,². Upon nociceptor activation and calcium-dependent exocytosis, CGRP is released from neurons, inducing sterile neurogenic inflammation involving arterial vasodilatation and plasma protein extravasation³^,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 migraine⁸^,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 evolution¹²^,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 treatment³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰. Some studies have found an elevation of CGRP in cohorts with episodic migraine relative to control participants¹⁰^,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 results¹⁶^,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 cardiologic²^,²⁵, dermatologic²⁶, obstetrical²⁷, rheumatologic²⁸^,²⁹, musculoskeletal³⁰^,³¹, endocrine³²^,³³, and viral diseases¹²^,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

Collect 5 mL of whole blood from the antecubital vein via standard venipuncture methods³⁴ into a 6 mL vacutainer ethylenediaminetetraacetic acid (EDTA) collection tube.
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.
Centrifuge the tube at 604 x g for 4 min at 4 °C within 60 min of blood collection.
Take off the plasma fraction with a pipette and transfer to 2 mL round-bottom sterile cryovials.
Immediately store the cryovials at -80 °C for up to 2 weeks.

2. Extraction of the plasma samples

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.
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.
Dilute 250 µL of plasma with 750 µL of 4% acetic acid.
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.
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.
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.
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.
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.
Place each tube in a mini centrifuge for 1 s to ensure that all the eluent is at the bottom of each tube.
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.
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

Add 250 µL of tris-buffered saline (TBS)/fish gelatin blocking buffer to each well on the plate.
Place a cover sheet over the plate and incubate for 2 h at RT.
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.
Dry the plate by leaving the plate in a laminar flow hood for 10 min.
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

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.
Thaw all samples and reagents to RT before performing the rest of the protocol.

5. Assay procedure

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.
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.
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).
Dispense 100 µL of EIA buffer into each NSB well.
Dispense 100 µL of the CGRP standards in duplicate into appropriate wells.
Dispense 100 µL of samples (reconstituted in EIA buffer) and quality control in duplicate into appropriate wells.
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.
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.
After incubation is completed, reconstitute the Ellman's reagent as per the kit instructions.
Invert the plate to remove all liquid. Rinse each well (as described above) three times with 300 µL of wash buffer.
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.
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.
Dispense 200 µL of Ellman's reagent into each well (not including the "Blank" wells).
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.
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.
Read the plate for absorbance at 405 nm (yellow color).

6. Data analysis

Calculate the average absorbance for each "Blank", NSB, standard, quality control, and the samples.
Subtract the NSB average absorbance values from the standard, quality control, and sample absorbance values.
Plot absorbance on the y-axis and concentration on the x-axis. Construct a standard curve using a four-parameter logistic regression model.
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).

Representative 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 humans²¹^,³⁵. If protease inhibitors are not used, and sample preparation takes longer than 60 min, then one can expect decreased levels of CGRP upon performing ELISA. Secondly, solid phase extraction prior to ELISA concentrates CGRP in the eluent and eliminates potentially interfering molecules from the plasma matrix. This extraction step increases the yield of CGRP in spike and recovery experiments (further discussed in the next section). Vacuum centrifugation in a cold room after extraction further limits the loss of CGRP. Thirdly, prior to ELISA, the microtiter plates must be blocked using a blocking buffer. This allows for the passive adsorption of non-specific proteins in the buffer to the remaining binding sites in the plates, thus reducing the background signal. Blocking helps reduce unrealistically high yields of CGRP.

Spike and recovery experiments determine if CGRP detection is affected by the differences between the standard curve diluent (here, EIA buffer) and experimental sample matrix (here, plasma). Plasma and/or serum may contain molecules that block antibody binding to CGRP or degrade the peptide, thus interfering with the assay's ability to detect and quantify the starting concentration of CGRP accurately. To this end, we added known amounts of CGRP to plasma samples-the "spiking" step-and calculated how much CGRP was "recovered" after extraction and ELISA. The CGRP stock was obtained from the manufacturer and stored in a -20 °C freezer until use.

In linearity of dilution testing, a sample is serially diluted, and concentrations of CGRP are calculated for each dilution. If the diluted samples do not exhibit appropriate linear decreases in CGRP concentration, this would indicate that the EIA buffer or plasma interferes with the detection of CGRP at some concentrations or that the standard curve is not accurate in the range that is affected. In performing the above-described spike and recovery experiment, we serially diluted the "spiked" samples; we spiked a plasma sample to give a concentration of CGRP of 200 pg/mL and subsequently diluted the sample to 100 pg/mL and then 50 pg/mL.

Plasma samples from three participants with no history of cardiovascular, respiratory, or recent headache or neurological symptoms were spiked with different concentrations of CGRP. The protocol was run for these samples, in addition to non-spiked control samples. Figure 1 shows an example of a plate map for spike and recovery and linearity of dilution experiments. A four-parameter logistic fit was performed to create a standard curve that allowed for fitting beyond the linear range (Table 1 and Figure 2). Recovery rates were calculated for each spiked sample and were within the acceptable range (Table 2). The linearity of dilution was demonstrated in the spiked samples (Figure 3). Table 3 shows results obtained from an unsuccessful spike and recovery experiment carried out following the manufacturer's instructions and prior to the inclusion of the added steps to block non-specific binding. These data show recovery rates exceeding the upper limit of 125%, indicating the presence of non-specific binding. After adding the steps in the protocol to block the plate using a blocking buffer (steps 3.1-3.5), we were able to reduce this effect and achieve acceptable recovery values (Table 2). In three patients without migraine or vestibular symptoms, we found that the average concentration of CGRP in plasma was 1.68 ± 0.13 pg/mL. Future experiments should aim to utilize the present methodology on a larger scale of control patients.

Figure 1: Example plate map. Each plate should contain standards, samples, blanks, and NSB wells, all in duplicate or triplicate. Blank wells should contain no liquid, and NSB wells should contain proprietary enzyme immunoassay buffer, enzyme-linked secondary antibody, and Ellman's reagent. The average absorbance values for the NSB wells should be subtracted from the absorbance values of the standard prior to creating the standard curve. The average absorbance values for the NSB wells should also be subtracted from the absorbance values of the samples, prior to calculating the percentage of recovery. There is no effect of plate position. Please click here to view a larger version of this figure.

Figure 2: Example of a four-parameter logistic (4PL) regression standard curve. This standard curve is mathematically described by an equation in a similar form to that seen in Table 1. A 4PL regression curve is fitter to biological systems compared to linear curves. This curve and equation are used to interpolate quality controls and samples on the same plate on which the standard was created. Please click here to view a larger version of this figure.

Figure 3: Linearity of dilution line chart showing samples spiked at 200 pg/mL, then serially diluted 1:2 and 1:4. This data demonstrate linearity over a wide range of dilutions, indicating the assay method is accurate across a wide range of CGRP concentrations. Please click here to view a larger version of this figure.

Parameter	Value
X₅₀	294.75
Equation
Equation form

Table 1: Example of afour-parameter logistic (4PL) regression standard curve equation with titers X₅₀. A 4PL regression curve accommodates for the complexity seen in biological systems. This curve was used to analyze the results of the ELISA, as it is more suitable than linear regression.

	Absorbance (OD)	Interpolated concentration (pg/mL)	Percent yield
Un-spiked control	-0.0434	Does not fall on curve (i.e., ~0)
Spiked at 200 pg/mL	0.2712	214.7 ± 23.4	107.40%
Spiked at 100 pg/mL	0.0966	102.7 ± 2.35	102.70%
Spiked at 50 pg/mL	0.0205	55.1 ± 12.65	110.20%

Table 2: Results of a successful spike and recovery experiment of three control participants due to a lack of solid phase extraction and plate blocking. The percentage of yield for spiked samples all fell within 20% of the ideal 100% yield, indicating that CGRP detection is not affected by the experimental sample matrix of plasma.

	Absorbance (OD)	Interpolated concentration (pg/mL)	Percent yield
Un-spiked control	-0.2087	Does not fall on curve (i.e., ~0)
Spiked at 200 pg/mL	2.371	264.2 ± 104	132.10%
Spiked at 100 pg/mL	1.134	167.5 ± 31.2	167.50%
Spiked at 50 pg/mL	0.3218	80.55 ± 4.54	161.10%

Table 3: Results of an unsuccessful spike and recovery experiment of three control participants due to a lack of plate blocking. The percentage of yield for spiked samples all fell well above the upper limit of 120% yield, indicating that the assay may be corrupted by NSB. After obtaining these results, we added steps 3.1-3.5 in the protocol to block NSB on the plate prior to the assay.

	Absorbance (OD)	Interpolated concentration (pg/mL)	Percent yield
Un-spiked control	0.0401	12.93
Spiked at 100 pg/mL	0.0315	10.17±2.31	10.17%
Spiked at 50 pg/mL	0.0152	4.895±15.4	9.79%
Spiked at 25 pg/mL	0.0007	0.2177±6.43	0.87%

Table 4: Results of an unsuccessful spike and recovery experiment of three control participants. The percentage of yield for spiked samples all fell well below the lower limit of 80% yield, indicating that further optimization was needed to address possible degradation and competitive binding processes. These results are from another manufacturer's competitive ELISA assay, with a lower limit of detection of 2.23 pg/mL, and intra-assay cv < 10% and inter-assay cv < 12%.

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). For reference, the widely accepted standard recovery is 80-120%³⁶. These results may suggest the presence of protease activity degrading CGRP²¹^,²²^,²³^,²⁴, other molecules binding to CGRP, thus limiting its availability for binding to the plate's antibodies, or competitive binding of non-target proteins to the plate's antibodies.

To control for these possible confounders, an extraction protocol was optimized to purify plasma through extraction with a proprietary sorbent prior to ELISA. This sorbent ostensibly isolates polar molecules such as CGRP. To determine the efficacy of extraction prior to ELISA, human plasma samples spiked with known concentrations of CGRP as positive controls underwent extraction and then ELISA. These spiked samples yielded only ~50%-60% recovery. These results indicated that exogenously added CGRP is not detected in plasma, as one would expect.

Various other steps of the protocol were optimized: adding serine protease inhibitor to whole blood samples prior to centrifugation to inhibit degradation²¹, lowering the temperature of vacuum centrifugation after extraction, and adjusting post-centrifugation resuspension methods. A spike and recovery experiment after these changes revealed unrealistically high CGRP concentrations, a greater than 120% recovery (Table 3). Reassuringly, higher than expected values were also recently reported after extraction using a similar assay by Messlinger et al.³⁷, indicating that this remains an issue. Messlinger et al. acknowledge that the assay after plasma extraction does not reproduce realistic CGRP concentrations, without providing a reason for this phenomenon³⁷.

We hypothesized that the residual binding capacity on the ELISA microtiter plates causing non-specific binding may be responsible for the high recovery rates. Prior to ELISA, the plate was incubated with a blocking buffer, TBS/fish gelatin buffer, to reduce this binding capacity. This additional step resulted in more appropriate recovery rates (Table 2). Thus, the modifications and optimizations discussed above, including the use of the blocking buffer, remain critical steps in the protocol. These steps have allowed for a lower limit of detection of 1.05 pg/mL, compared to that of the original manufacturer's kit listed as 2 pg/mL.

Regarding troubleshooting, step 2.10 calls for vacuum centrifugation to dry the eluent. The length of this step has been observed to vary depending on the type of microcentrifuge tube the samples are in. Using the microcentrifuge tubes described in the Table of Materials, this step usually lasts 5 h. However, when alternative microcentrifuge tubes of larger volumes are used, this step could last up to 11 h. An alternative to vacuum centrifugation may be lyophilization or freeze-drying the samples, which would require the samples to be frozen after extraction. However, lyophilization was not tested in the construction of this protocol. Additionally, if systematically lower than expected concentrations of CGRP are found, one should ensure that all reagents, especially the antibodies, are thawed to room temperature before use. The instability of reagents, such as the antibody and tracer solutions, could contribute to systematic error in binding CGRP. If yield continues to be inadequate, antibody binding to CGRP may be affected by the relative acidity of the eluent (methanol and acetic acid solution), despite reconstitution with a buffer. In this case, a pH rectification step would be a logical addition after reconstitution with buffer.

Limitations of this protocol include the limitations associated with any ELISA methodology, specifically antibody instability³⁸. Antibody reagents must be thawed to room temperature before use in the assay; however, warming for an extended period may cause denaturation and a consequently decreased effectiveness in binding CGRP. Additionally, CGRP may undergo degradation by a variety of non-serine proteases, leading to increased false negative results. Though this protocol limits degradation by adding a serine protease and limiting the sample processing time to less than 60 min, degradation may still occur. Another limitation is the lack of freeze-thaw cycles tested. Lee et al.³⁹ demonstrated that freeze-thaw cycles can increase or decrease serum and plasma protein quantities, depending on the susceptibility of the protein. The susceptibility of CGRP to freeze-thaw cycles has not been fully tested in the literature, though Messlinger et al. have noted that levels of CGRP decrease upon one cycle of freezing and thawing³⁷. Further, this protocol's yield may benefit from the use of protein LoBind tubes, designed with a hydrophilic surface, thus leading to improved protein recovery.

We note that these validation experiments did not appear to have been carried out by authors of recent studies quantifying CGRP in human plasma¹⁶^,¹⁸^,³⁵^,⁴⁰^,⁴¹. A review of the ELISA methodology by Messlinger et al.³⁷ emphasizes the need for such controls and calls into question studies that have not used them. We believe our work in validating and standardizing a protocol will put future CGRP research on a better footing. The implications of such a protocol are wide-ranging, and can theoretically be extended to investigating the biomarker status of CGRP in neurologic and non-neurologic disease processes²^,²⁵^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³.

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

We would like to thank Robert N. Cole, Lauren R. DeVine, and Marcos Iglesias for their helpful discussions regarding this protocol. This was supported in part by funding from the American Otological Society (Fellowship Grant, PSK), the American Hearing Research Foundation (90066548/90072266, JPC), and the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research (UL1 TR003098, NSF). The publication's contents are solely the responsibility of the authors and do not necessarily represent the official view of the Johns Hopkins ICTR, NCATS, or NIH.

Materials

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

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Krishnan, P. S., Zamuner, F. T., Jenks, C. M., Xie, J. Y., Zhang, L., Lehar, M., Fedarko, N. S., Brait, M., Carey, J. P. Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay. J. Vis. Exp. (196), e64775, doi:10.3791/64775 (2023).