TITLE: Detection and Quantification of Calcitonin Gene-Related Peptide in Human Blood Plasma using a Modified Enzyme-Linked Immunosorbent Assay
AUTHORS: Pavan S. Krishnan, Fernando T. Zamuner, Carolyn M. Jenks, Yanjun Xie, Lisa Zhang, Mohammed Lehar, Marcos Iglesias , Neal S. Fedarko, Mariana Brait, John P. Carey

[DESCRIPTION]
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. Here, we present a protocol for purification and quantification of CGRP in human plasma. This protocol involves sample collection and preparation, extraction as a means of purification, and quantification via enzyme-linked immunosorbent assay (ELISA). After finding unrealistically high CGRP concentrations in spike-and-recovery experiments using a commercial CGRP ELISA kit protocol, we modified the existing protocol to produce satisfactory percent recoveries. This protocol can theoretically be used to quantify CGRP concentrations in plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

[STEPS]
SECTION: Sample Collection and Preparation
1. Collect whole blood from antecubital vein into 6.0 mL BD Vacutainer EDTA collection tube 
SECTION: Sample Collection and Preparation
2. Add 0.5 mL of aprotinin competitive serine protease inhibitor to tube after collection
SECTION: Sample Collection and Preparation
3. Centrifuge at 3000 rpm for 4 minutes at 4°C, within 60 minutes of blood collection

SECTION: Sample Collection and Preparation
4. Take off plasma fraction with an Eppendorf pipette and transfer to 2 mL round bottom sterile cryovials
SECTION: Sample Collection and Preparation
5. Immediately store cryovials at -80°C for up to 2 weeks
SECTION: Extraction of Plasma Samples
6. Place an Oasis HLB Extraction cartridge inside a Falcon 15 mL conical centrifuge tube with the outer ridges of the cartridge supported by the outer ridges of the Falcon tube 
SECTION: Extraction of Plasma Samples
7. Activate the cartridge by first passing 5 mL of 100% methanol and then 10 mL of ultrapure water through the cartridge. As fluid is passed through the cartridge, it will collect in the Falcon tube. Throw out excess fluid as it accumulates to ensure that the fluid does not engulf the cartridge. Ensure that the cartridge does not dry during the course of the experiment.
SECTION: Extraction of Plasma Samples
8. Dilute blood plasma at 1:4 with 4% acetic acid 
SECTION: Extraction of Plasma Samples
8.1. Please note: The above ratio refers to dilution factor NOT dilution ratio (e.g. 1:4 refers to 1 volume of solute in 4 volumes of whole solution). 

Per the manufacturer, each cartridge becomes saturated with 1 mL of blood plasma and acetic acid solution, therefore we dilute 250 uL of blood plasma with 750 uL of acetic acid. Theoretically, if one chooses to use more than 250 uL of blood plasma, one would need to dilute with the appropriate amount of acetic acid and pass this solution through more than one cartridge.
SECTION: Extraction of Plasma Samples
9. Pass 1 mL of blood plasma and acetic acid solution slowly through the cartridge at 1 mL per 30 seconds
SECTION: Extraction of Plasma Samples
10. Wash the cartridge with 10 mL of 4% acetic acid. As done previously, throw out excess fluid as it accumulates to ensure that the fluid does not engulf the cartridge.

SECTION: Extraction of Plasma Samples
11. After the last drop of acetic acid is released from the cartridge, remove the cartridge from the Falcon tube and place in a new Falcon 15 mL conical centrifuge tube. 
SECTION: Extraction of Plasma Samples
12. Prepare a 3 mL solution of 100% methanol and 4% acetic acid (10:1) and elute the CGRP by passing this solution through the cartridge 1 mL at a time. Pause for 25 minutes between each mL of solution passed. Do NOT throw out the eluent.
SECTION: Extraction of Plasma Samples
12.1. Please note: Per the definition of dilution factor, the 3 mL solution should contain 2.7 mL of 4% acetic acid and 0.3 mL of 100% methanol.
SECTION: Extraction of Plasma Samples
13. 25 minutes after the last mL of methanol and acetic acid solution is passed, you should find 3 mL of eluent in the Falcon tube. Place each 1 mL of eluent into a 1.7 mL Safeseal microcentrifuge tube. Thus, you will have 3 tubes from each cartridge.
SECTION: Extraction of Plasma Samples
14. Place each tube in a mini centrifuge for 1 second to ensure that all eluent is at the bottom of each tube.
SECTION: Extraction of Plasma Samples
15. Dry all samples by vacuum centrifugation
SECTION: Extraction of Plasma Samples
15.1. Please note: Time taken by the vacuum centrifuge to dry samples will depend on the type of eppendorf tube used and the concentrator. For example, the vacuum centrifugation step lasted 5 hours using the aforementioned Safeseal tubes in a Vacufuge plus - Centrifuge Concentrator. Using alternative eppendorf tubes in the same centrifuge caused the centrifuge step to last up to 11 hours.
SECTION: Extraction of Plasma Samples
16. Reconstitute the dried sample with a volume of EIA buffer equal to the original sample volume. EIA buffer should be previously warmed to 37°C.
SECTION: Extraction of Plasma Samples
16.1. Example: If using 250 uL of original sample (before diluting with acetic acid), reconstitute with 250 uL of EIA buffer. 
SECTION: Plate Coating
17. Add 250 uL of Bioworld TBS/Fish Gelatin blocking buffer to each well on the plate.
SECTION: Plate Coating
18. Place a cover sheet over the plate and incubate for 2 hours at room temperature.
SECTION: Plate Coating
19. Remove cover sheet and empty the plate by inverting it. Then blot the plate on a paper towel to discard any trace of liquid.
SECTION: Plate Coating
20. Dry the plate by leaving the plate in a laminar flow hood for 10 minutes
SECTION: Plate Coating
21. After the plates are visibly dry, place the plates in a grip-sealed foil pouch with a silica gel dessicant sachet and store the pouch at -20°C.
SECTION: Prior to Assay Procedure
22. Prepare reagents (EIA buffer, CGRP standard, CGRP quality control, CGRP tracer, wash buffer) per the kit instructions. Prepare Ellman's reagent only after 16-20 hour incubation period ends. 
SECTION: Prior to Assay Procedure
23. Let all samples and reagents reach room temperature before performing the assay
SECTION: Assay Procedure
24. Rinse each well in the blocked plate 5 times with wash buffer (300 uL/well). For each rinse, pause 30 seconds after placing the wash buffer in the wells, then throw out the liquid.
SECTION: Assay Procedure
25. After the final rinse, remove all buffer from the wells by inverting the plate and blotting the last drops onto a paper towel. Tap the inverted plate until all liquid is visibly removed from the wells.

SECTION: Assay Procedure
26. Set aside at least 2 wells with no reagents or buffer to be known as "Blank" wells. Then set aside at least 2 other wells for non-specific binding (NSB).
SECTION: Assay Procedure
27. Dispense 100 uL of EIA buffer into each NSB well. 
SECTION: Assay Procedure
28. Dispense 100 uL of the CGRP standards in duplicate to appropriate wells.
SECTION: Assay Procedure
29. Dispense 100 uL of samples (reconstituted in EIA buffer) and quality control in duplicate to appropriate wells.
SECTION: Assay Procedure
30. Dispense 100 uL of CGRP tracer to each well containing NSB, CGRP standards, samples, and quality control. Do not dispense tracer to the wells dedicated as "Blank."
SECTION: Assay Procedure
31. Cover the plate with a cover sheet and incubate for 16-20 hours at 4°C.
SECTION: Assay Procedure
32. After incubation is completed, reconstitute Ellman's reagent per kit instructions.
SECTION: Assay Procedure
33. Invert the plate to remove all liquid. Rinse each well (as described above) 3 times with 300 uL of wash buffer. 
SECTION: Assay Procedure
34. After 3rd rinse, remove the liquid from the plates, and place the plate on a shaker plate and shake for 2 minutes. 
SECTION: Assay Procedure
35. Wash the plate an additional 3 times. After the final rinse, remove all buffer from the wells by inverting the plate and blotting the last drops onto a paper towel. Tap the inverted plate until all liquid is visibly removed from the wells.

SECTION: Assay Procedure
36. Dispense 200 uL of Ellman's reagent into each well (not including the "Blank" wells). 
SECTION: Assay Procedure
37. Cover the plate with a new cover sheet and wrap in aluminum foil to prevent any light exposure. Incubate in the dark at room temperature for 1 hour. 
SECTION: Assay Procedure
38. Wipe the bottom of the plate with a paper towel to ensure that there is no liquid outside of the wells.
SECTION: Assay Procedure
39. Read the plate for Absorbance at 405nm (yellow color)
SECTION: Data Analysis
40. Calculate average absorbance for each "Blank," NSB, standard, quality control, and samples. 


SECTION: Data Analysis
41. Subtract "Blank" and NSB average absorbance values from standard, quality control, and sample absorbance values
SECTION: Data Analysis
42. Plot absorbance on the y-axis and concentration on the x-axis.  Construct a standard curve using a Four-Parameter Logistic regression model.
SECTION: Data Analysis
43. 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.
SECTION: Data Analysis
43.1. The standard curve is validated only if the calculated interpolated concentrations for the quality control is within 25% of the expected concentration (usually 125 pg/ml, but see label of the quality control vial).