We would like to thank Robert N. Cole, Lauren R. DeVine, and Marcos Iglesias for their helpful discussions regarding this protocol.&#160;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&#39;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.

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
<ol>
	<li>Collect 5 mL of whole blood from the antecubital vein via standard venipuncture methods34 into a 6 mL vacutainer ethylenediaminetetraacetic acid (EDTA) collection tube.</li>
	<li>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.</li>
	<li>Centrifuge the tube at 604 x g for 4 min at 4 &#176;C within 60 min of blood collection.</li>
	<li>Take off the plasma fraction with a pipette and transfer to 2 mL round-bottom sterile cryovials.</li>
	<li>Immediately store the cryovials at -80 &#176;C for up to 2 weeks.</li>
</ol>
2. Extraction of the plasma samples
<ol>
	<li>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.</li>
	<li>Activate the cartridge by first passing 5 mL of 100% methanol and then 10 mL of ultrapure water through the cartridge.
	<ol>
		<li>As fluid is passed through the cartridge via gravity-driven flow, it will collect in the tube.</li>
		<li>Throw out excess fluid as it accumulates to ensure that the fluid does not engulf the cartridge.</li>
		<li>Ensure that the cartridge does not dry during the course of the experiment.</li>
	</ol>
	</li>
	<li>Dilute 250 &#181;L of plasma with 750 &#181;L of 4% acetic acid.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place each tube in a mini centrifuge for 1 s to ensure that all the eluent is at the bottom of each tube.</li>
	<li>Dry all the samples by vacuum centrifugation at 4 &#176;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.</li>
	<li>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 &#176;C. 
	&#8203;NOTE: The volume of EIA buffer used to reconstitute the dried sample should be equal to the original sample volume, or 250 &#181;L.</li>
</ol>
3. Preparation of the ELISA plate - blocking to limit non-specific binding
<ol>
	<li>Add 250 &#181;L of tris-buffered saline (TBS)/fish gelatin blocking buffer to each well on the plate.</li>
	<li>Place a cover sheet over the plate and incubate for 2 h at RT.</li>
	<li>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.</li>
	<li>Dry the plate by leaving the plate in a laminar flow hood for 10 min.</li>
	<li>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 &#176;C for 24 h. 
	&#8203;NOTE: The plates should be used within 4 weeks of plate incubation.</li>
</ol>
4. Prior to the assay procedure
<ol>
	<li>Prepare reagents (EIA buffer, CGRP standard, CGRP quality control, CGRP tracer, wash buffer) as per the kit instructions. Prepare Ellman&#39;s reagent only after the 16-20 h incubation period ends.</li>
	<li>Thaw all samples and reagents to RT before performing the rest of the protocol.</li>
</ol>
5. Assay procedure
<ol>
	<li>Rinse each well in the blocked plate five times with a kit-provided wash buffer (300 &#181;L/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.</li>
	<li>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.</li>
	<li>Set aside at least two wells with no reagents or buffer to be known as &#34;Blank&#34; wells. Then, set aside at least two other wells for non-specific binding (NSB).</li>
	<li>Dispense 100 &#181;L of EIA buffer into each NSB well.</li>
	<li>Dispense 100 &#181;L of the CGRP standards in duplicate into appropriate wells.</li>
	<li>Dispense 100 &#181;L of samples (reconstituted in EIA buffer) and quality control in duplicate into appropriate wells.</li>
	<li>Dispense 100 &#181;L 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 &#34;Blank&#34; wells.</li>
	<li>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 &#176;C.</li>
	<li>After incubation is completed, reconstitute the Ellman&#39;s reagent as per the kit instructions.</li>
	<li>Invert the plate to remove all liquid. Rinse each well (as described above) three times with 300 &#181;L of wash buffer.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Dispense 200 &#181;L of Ellman&#39;s reagent into each well (not including the &#34;Blank&#34; wells).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Read the plate for absorbance at 405 nm (yellow color).</li>
</ol>
6. Data analysis
<ol>
	<li>Calculate the average absorbance for each &#34;Blank&#34;, NSB, standard, quality control, and the samples.</li>
	<li>Subtract the NSB average absorbance values from the standard, quality control, and sample absorbance values.</li>
	<li>Plot absorbance on the y-axis and concentration on the x-axis. Construct a standard curve using a four-parameter logistic regression model.</li>
	<li>Once the curve is constructed, use the curve&#39;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).</li>
</ol>

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<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((WHO+Guidelines+on+Drawing+Blood%3A+Best+Practices+in+Phlebotomy.+3%2C+Blood-sampling+systems%5BTitle%5D)+AND+%22World+Health+Organization%22%5BJournal%5D)">WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. 3, Blood-sampling systems.</a> World Health Organization. , Geneva. (2010).</li>
<li>Raffaelli, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+calcitonin+gene-related+peptide+%28CGRP%29+in+migraine+and+endometriosis+during+the+menstrual+cycle%5BTitle%5D)+AND+%22Annals+of+Clinical+and+Translational+Neurology%22%5BJournal%5D)">Plasma calcitonin gene-related peptide (CGRP) in migraine and endometriosis during the menstrual cycle.</a> Annals of Clinical and Translational Neurology. 8 (6), 1251-1259 (2021).</li>
<li>Andreasson, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+guide+to+immunoassay+method+validation%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">A practical guide to immunoassay method validation.</a> Frontiers in Neurology. 6, 179(2015).</li>
<li>Messlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CGRP+measurements+in+human+plasma-a+methodological+study%5BTitle%5D)+AND+%22Cephalalgia%3A+An+International+Journal+of+Headache%22%5BJournal%5D)">CGRP measurements in human plasma-a methodological study.</a> Cephalalgia: An International Journal of Headache. 41 (13), 1359-1373 (2021).</li>
<li>Sakamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme-linked+immunosorbent+assay+for+the+quantitative%2Fqualitative+analysis+of+plant+secondary+metabolites%5BTitle%5D)+AND+%22Journal+of+Natural+Medicines%22%5BJournal%5D)">Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.</a> Journal of Natural Medicines. 72 (1), 32-42 (2018).</li>
<li>Lee, J. -E., Kim, S. Y., Shin, S. -Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+repeated+freezing+and+thawing+on+biomarker+stability+in+plasma+and+serum+samples%5BTitle%5D)+AND+%22Osong+Public+Health+and+Research+Perspectives%22%5BJournal%5D)">Effect of repeated freezing and thawing on biomarker stability in plasma and serum samples.</a> Osong Public Health and Research Perspectives. 6 (6), 357-362 (2015).</li>
<li>Fan, P. -C., Kuo, P. -H., Lee, M. T., Chang, S. -H., Chiou, L. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+calcitonin+gene-related+peptide%3A+A+potential+biomarker+for+diagnosis+and+therapeutic+responses+in+pediatric+migraine%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">Plasma calcitonin gene-related peptide: A potential biomarker for diagnosis and therapeutic responses in pediatric migraine.</a> Frontiers in Neurology. 10, 10(2019).</li>
<li>Raffaelli, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Change+of+CGRP+plasma+concentrations+in+migraine+after+discontinuation+of+CGRP-%28receptor%29+monoclonal+antibodies%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Change of CGRP plasma concentrations in migraine after discontinuation of CGRP-(receptor) monoclonal antibodies.</a> Pharmaceutics. 15 (1), 293(2023).</li>
</ol>

The authors have no further disclosures to add.

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</...

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, &#945;- and &#946;-CGRP, share more than 90% homology and share physiologic functions; however, &#945;CGRP is found in the central and peripheral nervous system, while &#946;CGRP 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&#39;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 &#945;- and &#946;CGRP in human plasma. The kit&#39;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.

<table><tbody><tr><td>1.7 mL Safeseal microcentrifuge tube</td><td>Sorenson Bioscience, Inc.</td><td>11510</td><td></td></tr><tr><td>99% methanol</td><td>ThermoFisher Scientific</td><td>L13255.0F</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Falcon</td><td>14-959-49B</td><td></td></tr><tr><td>2 mL round bottom sterile cryovials</td><td>CRYO.S</td><td>122263</td><td></td></tr><tr><td>4% acetic acid</td><td>ThermoFisher Scientific</td><td>035572.K2</td><td></td></tr><tr><td>6.0 mL Vacutainer EDTA collection tube</td><td>BD</td><td>367863</td><td></td></tr><tr><td>Allegra 64R benchtop centrifuge</td><td>Beckman Coulter, Inc.</td><td>367586</td><td></td></tr><tr><td>Aprotinin</td><td>VWR</td><td>76344-814</td><td></td></tr><tr><td>CGRP (human) ELISA kit</td><td>Bertin Bioreagent</td><td>A05481</td><td></td></tr><tr><td>CGRP stock</td><td>Bertin Bioreagent</td><td></td><td></td></tr><tr><td>EIA Buffer</td><td>Bertin Bioreagent</td><td>A07000</td><td></td></tr><tr><td>Ellman&#039;s Reagent</td><td>Bertin Bioreagent</td><td>A09000_49+1</td><td></td></tr><tr><td>Multichannel pipettes</td><td>ThermoFisher Scientific</td><td>4661180N</td><td></td></tr><tr><td>Oasis HLB 3 cc Vac Cartridges</td><td>Waters</td><td>WAT094226</td><td></td></tr><tr><td>Orbital Shaker</td><td>Bellco</td><td>7744-01010</td><td></td></tr><tr><td>Precision micropipettes</td><td>ThermoFisher Scientific</td><td>F144055MG</td><td></td></tr><tr><td>SpectraMax M Series Multi-Mode Microplate reader</td><td>Molecular Devices</td><td>Part Number M2</td><td></td></tr><tr><td>TBS/Fish Gelatin</td><td>Bioworld, from Fischer Scientific</td><td>50-199-167</td><td></td></tr><tr><td>Ultrapure water ELISA Grade</td><td>Bertin Bioreagent</td><td>A07001</td><td></td></tr><tr><td>Vacufuge plus - Centrifuge Concentrator</td><td>Eppendorf</td><td>22820109</td><td></td></tr><tr><td>Wash Buffer</td><td>Bertin Bioreagent</td><td>A17000</td><td></td></tr></tbody></table>

detection and quantification of calcitonin gene-related peptide (cgrp) in human plasma using a modified enzyme-linked immunosorbent assay

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.

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 m...

Watch this Scientific Journal Video about Detection and Quantification of Calcitonin Gene-Related Peptide CGRP in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay at JoVE.com

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

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.

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

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a ...

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Research

JoVE Journal

Biochemistry

2.5K Views.  Johns Hopkins University School of Medicine. Dit gemodificeerde ELISA-protocol maakt de zuivering en nauwkeurige kwantificering van CGRP in menselijk plasma mogelijk. Het heeft de nodige validatie-experimenten ondergaan en kan dienen om toekomstige kwantificeringsmethoden te standaardiseren. Dit protocol biedt de voordelen van het opnemen van monsterverwerking vóór significante proteaseafbraak, zuivering via vastefase-extractie en beperking van niet-specifieke binding door toepassing van blokkerende buffer op de microtiterplaten.

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

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Bioengineering

Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, especially in breast cancer and other HER2-expressing cancer types. In the clinic, the gold-standard immunohistochemical diagnostic methods employing the specific anti-HER2 antibodies are used to measure the expression level of the membrane-bound receptor. The soluble extracellular domain (ECD) of HER2 that is released from the overexpressing cells circulates in the blood and can reflect the tissue expression of the receptor. There is a need for accurate and validated assays to correlate the concentration of the circulating HER2 protein with disease clinical manifestations.
Our team has developed and validated the novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the membrane-bound and the released from cells ECD domain of HER2. The assay uses two unique monoclonal antibodies specific to HER2 developed previously. The quantitation range includes HER2 concentration from 1.56-100 ng/mL, which is expected for cancer cells cultured in vitro and shows sensitivity at the level of 0.5 ng/mL. The satisfactory intra- and inter-assay precision and accuracy of the method make it applicable for HER2 quantification in various types of biological samples, including cell culture medium, serum, and solid tumor tissue. Here, we focus on the comprehensive determination of the receptor-associated and secreted by the in vitro cultured cancer cells. The paper presents a step-by-step protocol for the quantification of HER2 protein that can be employed for testing a variety of cell lines, blood, and tissues.

We present a validated sandwich ELISA assay using novel anti-HER2 monoclonal antibodies. This assay enables precise quantification of cell-bound and released HER2 protein from in vitro cultured cells and other samples, including blood and tissues.

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, ...

Cancer Research

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Medicine

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Neuroscience

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Biology

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid hormone (PTH), which leads to high serum phosphorus levels and low serum calcium levels. HypoPT most commonly results from accidental damage to the glands or their removal during thyroid or other anterior neck surgery. Parathyroid/thyroid surgery has become more common in recent years, with a corresponding rise in the occurrence of HypoPT as a postoperative complication. There is a critical need for a HypoPT animal model to better understand the mechanisms underlying the effects of HypoPT on mineral ion homeostasis and to verify the therapeutic effectiveness of novel treatments. Here, a technique is reported to create acquired HypoPT in male rats by performing parathyroidectomy (PTX) using carbon nanoparticles. The rat model shows great promise over the mouse models of hypoparathyroidism.&#160;Importantly, the human PTH receptor binding region has an 84.2% sequence similarity with that of the rat, which is higher than the 73.7% similarity shared with mice. Moreover, the effects of estrogen, which can affect the PTH/PTHrP receptor signaling pathway, have not been fully investigated in male rats. Carbon nanoparticles are lymphatic tracers that stain the thyroid lymph nodes black without affecting their function, but they do not stain the parathyroid glands, which makes them easy to identify and remove. In this study, serum PTH levels were undetectable after PTX, and this resulted in significant hypocalcemia and hyperphosphatemia. Thus, the clinical state of postoperative HypoPT can be remarkably represented in the rat model. Carbon-nanoparticle-assisted PTX can, therefore, serve as an extraordinarily effective and readily implementable model for studying the pathogenesis, treatment, and prognosis of HypoPT.

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

An animal model of acquired hypoparathyroidism (HypoPT) is crucial to understanding how HypoPT affects mineral ion homeostasis and to verify the effectiveness of novel treatments. Here, a technique is presented to generate an acquired hypoparathyroidism (AHypoPT) rat model by parathyroidectomy (PTX) using carbon nanoparticles.

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid ...

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Source: Gu, Y. et al., <a href="https://app.jove.com/v/57227">Electrochemiluminescence Assays for Human Islet Autoantibodies.</a>&#160;J. Vis. Exp. (2018)
This video showcases the electrochemiluminescence detection of islet autoantibodies in human serum. The antibodies in the serum bridge the ruthenium-conjugated antigen to the biotinylated capture antigen, forming complexes that are detected by electrochemiluminescence measurements.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Buffer Preparation

	Labeling ...

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

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