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

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 adequatel....

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