172 lines
55 KiB
Text
172 lines
55 KiB
Text
<!DOCTYPE html>
|
|
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" class="no-js no-jr">
|
|
<head>
|
|
<!-- For pinger, set start time and add meta elements. -->
|
|
<script type="text/javascript">var ncbi_startTime = new Date();</script>
|
|
|
|
<!-- Logger begin -->
|
|
<meta name="ncbi_db" content="books">
|
|
<meta name="ncbi_pdid" content="book-part">
|
|
<meta name="ncbi_acc" content="NBK604953">
|
|
<meta name="ncbi_domain" content="nciprotocols">
|
|
<meta name="ncbi_report" content="reader">
|
|
<meta name="ncbi_type" content="fulltext">
|
|
<meta name="ncbi_objectid" content="">
|
|
<meta name="ncbi_pcid" content="/NBK604953/?report=reader">
|
|
<meta name="ncbi_pagename" content="Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf">
|
|
<meta name="ncbi_bookparttype" content="chapter">
|
|
<meta name="ncbi_app" content="bookshelf">
|
|
<!-- Logger end -->
|
|
|
|
<!--component id="Page" label="meta"/-->
|
|
<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf</title>
|
|
<meta charset="utf-8">
|
|
<meta name="apple-mobile-web-app-capable" content="no">
|
|
<meta name="viewport" content="initial-scale=1,minimum-scale=1,maximum-scale=1,user-scalable=no">
|
|
<meta name="jr-col-layout" content="auto">
|
|
<meta name="jr-prev-unit" content="/books/n/nciprotocols/ncipr65/?report=reader">
|
|
<meta name="jr-next-unit" content="/books/n/nciprotocols/ncipr62/?report=reader">
|
|
<meta name="bk-toc-url" content="/books/n/nciprotocols/?report=toc">
|
|
<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE">
|
|
<meta name="citation_inbook_title" content="National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]">
|
|
<meta name="citation_title" content="Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants">
|
|
<meta name="citation_publisher" content="National Cancer Institute (US)">
|
|
<meta name="citation_date" content="2020/09">
|
|
<meta name="citation_author" content="Edward Cedrone">
|
|
<meta name="citation_author" content="Timothy M. Potter">
|
|
<meta name="citation_author" content="Barry W. Neun">
|
|
<meta name="citation_author" content="Abby Tyler">
|
|
<meta name="citation_author" content="Marina A. Dobrovolskaia">
|
|
<meta name="citation_pmid" content="39012981">
|
|
<meta name="citation_doi" content="10.17917/P2T3-8P84">
|
|
<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK604953/">
|
|
<link rel="schema.DC" href="http://purl.org/DC/elements/1.0/">
|
|
<meta name="DC.Title" content="Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants">
|
|
<meta name="DC.Type" content="Text">
|
|
<meta name="DC.Publisher" content="National Cancer Institute (US)">
|
|
<meta name="DC.Contributor" content="Edward Cedrone">
|
|
<meta name="DC.Contributor" content="Timothy M. Potter">
|
|
<meta name="DC.Contributor" content="Barry W. Neun">
|
|
<meta name="DC.Contributor" content="Abby Tyler">
|
|
<meta name="DC.Contributor" content="Marina A. Dobrovolskaia">
|
|
<meta name="DC.Date" content="2020/09">
|
|
<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK604953/">
|
|
<meta name="description" content="Cytokines, chemokines and interferons (IFN) are soluble mediators of inflammation and a variety of responses that orchestrate both innate and adaptive immunity in health and disease [1–10]. Understanding the induction of these biomarkers in response to a drug product helps to establish the product’s safety profile and shed light on the mechanism of action or mechanism of toxicity. This document describes experimental procedure for analysis of culture supernatants by multiplex ELISA to detect presence of type I IFNs (IFNα, IFNβ, IFNω), type II IFN (IFNγ), type III IFN (IFNλ), cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-21, IL-22, IL-23, IL-27, IP-10, MCP-1, MCP-2, MIP-1α, MIP-1β, RANTES, TNFα) in culture supernatants. NCL protocol ITA-10 should be referred to for details of preparation of culture supernatants. Protocols for single-plex ELISA (ITA-22, ITA-23, ITA-24 and ITA-25) can also be considered. The advantage of this protocol over single-plex ELISA is that it allows simultaneous analysis of a broad spectrum of markers.">
|
|
<meta name="og:title" content="Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants">
|
|
<meta name="og:type" content="book">
|
|
<meta name="og:description" content="Cytokines, chemokines and interferons (IFN) are soluble mediators of inflammation and a variety of responses that orchestrate both innate and adaptive immunity in health and disease [1–10]. Understanding the induction of these biomarkers in response to a drug product helps to establish the product’s safety profile and shed light on the mechanism of action or mechanism of toxicity. This document describes experimental procedure for analysis of culture supernatants by multiplex ELISA to detect presence of type I IFNs (IFNα, IFNβ, IFNω), type II IFN (IFNγ), type III IFN (IFNλ), cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-21, IL-22, IL-23, IL-27, IP-10, MCP-1, MCP-2, MIP-1α, MIP-1β, RANTES, TNFα) in culture supernatants. NCL protocol ITA-10 should be referred to for details of preparation of culture supernatants. Protocols for single-plex ELISA (ITA-22, ITA-23, ITA-24 and ITA-25) can also be considered. The advantage of this protocol over single-plex ELISA is that it allows simultaneous analysis of a broad spectrum of markers.">
|
|
<meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK604953/">
|
|
<meta name="og:site_name" content="NCBI Bookshelf">
|
|
<meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-nciprotocols-lrg.png">
|
|
<meta name="twitter:card" content="summary">
|
|
<meta name="twitter:site" content="@ncbibooks">
|
|
<meta name="bk-non-canon-loc" content="/books/n/nciprotocols/ncipr64/?report=reader">
|
|
<link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK604953/">
|
|
<link href="https://fonts.googleapis.com/css?family=Archivo+Narrow:400,700,400italic,700italic&subset=latin" rel="stylesheet" type="text/css">
|
|
<link rel="stylesheet" href="/corehtml/pmc/jatsreader/ptpmc_3.22/css/libs.min.css">
|
|
<link rel="stylesheet" href="/corehtml/pmc/jatsreader/ptpmc_3.22/css/jr.min.css">
|
|
<meta name="format-detection" content="telephone=no">
|
|
<link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css">
|
|
<link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css//books_print.min.css" type="text/css" media="print">
|
|
<link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_reader.min.css" type="text/css">
|
|
<style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style>
|
|
|
|
<link rel="shortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico">
|
|
<meta name="ncbi_phid" content="CE8CBB6B7C8E176100000000011000FB.m_5">
|
|
<meta name='referrer' content='origin-when-cross-origin'/><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3852956/3849091.css"></head>
|
|
<body>
|
|
<!-- Book content! -->
|
|
|
|
|
|
<div id="jr" data-jr-path="/corehtml/pmc/jatsreader/ptpmc_3.22/"><div class="jr-unsupported"><table class="modal"><tr><td><span class="attn inline-block"></span><br />Your browser does not support the NLM PubReader view.<br />Go to <a href="/pmc/about/pr-browsers/">this page</a> to see a list of supported browsers<br />or return to the <br /><a href="/books/NBK604953/?report=classic">regular view</a>.</td></tr></table></div><div id="jr-ui" class="hidden"><nav id="jr-head"><div class="flexh tb"><div id="jr-tb1"><a id="jr-links-sw" class="hidden" title="Links"><svg xmlns="http://www.w3.org/2000/svg" version="1.1" x="0px" y="0px" viewBox="0 0 70.6 85.3" style="enable-background:new 0 0 70.6 85.3;vertical-align:middle" xml:space="preserve" width="24" height="24">
|
|
<style type="text/css">.st0{fill:#939598;}</style>
|
|
<g>
|
|
<path class="st0" d="M36,0C12.8,2.2-22.4,14.6,19.6,32.5C40.7,41.4-30.6,14,35.9,9.8"></path>
|
|
<path class="st0" d="M34.5,85.3c23.2-2.2,58.4-14.6,16.4-32.5c-21.1-8.9,50.2,18.5-16.3,22.7"></path>
|
|
<path class="st0" d="M34.7,37.1c66.5-4.2-4.8-31.6,16.3-22.7c42.1,17.9,6.9,30.3-16.4,32.5h1.7c-66.2,4.4,4.8,31.6-16.3,22.7 c-42.1-17.9-6.9-30.3,16.4-32.5"></path>
|
|
</g>
|
|
</svg> Books</a></div><div class="jr-rhead f1 flexh"><div class="head"><a href="/books/n/nciprotocols/ncipr65/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="body"><div class="t">NCL Method ITA-27, Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants: Version 1</div><div class="j">National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]</div></div><div class="tail"><a href="/books/n/nciprotocols/ncipr62/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></div><div id="jr-tb2"><a id="jr-bkhelp-sw" class="btn wsprkl hidden" title="Help with NLM PubReader">?</a><a id="jr-help-sw" class="btn wsprkl hidden" title="Settings and typography in NLM PubReader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512" preserveAspectRatio="none"><path d="M462,283.742v-55.485l-29.981-10.662c-11.431-4.065-20.628-12.794-25.274-24.001 c-0.002-0.004-0.004-0.009-0.006-0.013c-4.659-11.235-4.333-23.918,0.889-34.903l13.653-28.724l-39.234-39.234l-28.72,13.652 c-10.979,5.219-23.68,5.546-34.908,0.889c-0.005-0.002-0.01-0.003-0.014-0.005c-11.215-4.65-19.933-13.834-24-25.273L283.741,50 h-55.484l-10.662,29.981c-4.065,11.431-12.794,20.627-24.001,25.274c-0.005,0.002-0.009,0.004-0.014,0.005 c-11.235,4.66-23.919,4.333-34.905-0.889l-28.723-13.653l-39.234,39.234l13.653,28.721c5.219,10.979,5.545,23.681,0.889,34.91 c-0.002,0.004-0.004,0.009-0.006,0.013c-4.649,11.214-13.834,19.931-25.271,23.998L50,228.257v55.485l29.98,10.661 c11.431,4.065,20.627,12.794,25.274,24c0.002,0.005,0.003,0.01,0.005,0.014c4.66,11.236,4.334,23.921-0.888,34.906l-13.654,28.723 l39.234,39.234l28.721-13.652c10.979-5.219,23.681-5.546,34.909-0.889c0.005,0.002,0.01,0.004,0.014,0.006 c11.214,4.649,19.93,13.833,23.998,25.271L228.257,462h55.484l10.595-29.79c4.103-11.538,12.908-20.824,24.216-25.525 c0.005-0.002,0.009-0.004,0.014-0.006c11.127-4.628,23.694-4.311,34.578,0.863l28.902,13.738l39.234-39.234l-13.66-28.737 c-5.214-10.969-5.539-23.659-0.886-34.877c0.002-0.005,0.004-0.009,0.006-0.014c4.654-11.225,13.848-19.949,25.297-24.021 L462,283.742z M256,331.546c-41.724,0-75.548-33.823-75.548-75.546s33.824-75.547,75.548-75.547 c41.723,0,75.546,33.824,75.546,75.547S297.723,331.546,256,331.546z"></path></svg></a><a id="jr-fip-sw" class="btn wsprkl hidden" title="Find"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 550 600" preserveAspectRatio="none"><path fill="none" stroke="#000" stroke-width="36" stroke-linecap="round" style="fill:#FFF" d="m320,350a153,153 0 1,0-2,2l170,170m-91-117 110,110-26,26-110-110"></path></svg></a><a id="jr-rtoc-sw" class="btn wsprkl hidden" title="Table of Contents"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M20,20h10v8H20V20zM36,20h44v8H36V20zM20,37.33h10v8H20V37.33zM36,37.33h44v8H36V37.33zM20,54.66h10v8H20V54.66zM36,54.66h44v8H36V54.66zM20,72h10v8 H20V72zM36,72h44v8H36V72z"></path></svg></a></div></div></nav><nav id="jr-dash" class="noselect"><nav id="jr-dash" class="noselect"><div id="jr-pi" class="hidden"><a id="jr-pi-prev" class="hidden" title="Previous page"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a><div class="pginfo">Page <i class="jr-pg-pn">0</i> of <i class="jr-pg-lp">0</i></div><a id="jr-pi-next" class="hidden" title="Next page"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div><div id="jr-is-tb"><a id="jr-is-sw" class="btn wsprkl hidden" title="Switch between Figures/Tables strip and Progress bar"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><rect x="10" y="40" width="20" height="20"></rect><rect x="40" y="40" width="20" height="20"></rect><rect x="70" y="40" width="20" height="20"></rect></svg></a></div><nav id="jr-istrip" class="istrip hidden"><a id="jr-is-prev" href="#" class="hidden" title="Previous"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M80,40 60,65 80,90 70,90 50,65 70,40z M50,40 30,65 50,90 40,90 20,65 40,40z"></path><text x="35" y="25" textLength="60" style="font-size:25px">Prev</text></svg></a><a id="jr-is-next" href="#" class="hidden" title="Next"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M20,40 40,65 20,90 30,90 50,65 30,40z M50,40 70,65 50,90 60,90 80,65 60,40z"></path><text x="15" y="25" textLength="60" style="font-size:25px">Next</text></svg></a></nav><nav id="jr-progress"></nav></nav></nav><aside id="jr-links-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">NCBI Bookshelf</div></div><div class="cnt lol f1"><a href="/books/">Home</a><a href="/books/browse/">Browse All Titles</a><a class="btn share" target="_blank" rel="noopener noreferrer" href="https://www.facebook.com/sharer/sharer.php?u=https://www.ncbi.nlm.nih.gov/books/NBK604953/"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 33 33" style="vertical-align:middle" width="24" height="24" preserveAspectRatio="none"><g><path d="M 17.996,32L 12,32 L 12,16 l-4,0 l0-5.514 l 4-0.002l-0.006-3.248C 11.993,2.737, 13.213,0, 18.512,0l 4.412,0 l0,5.515 l-2.757,0 c-2.063,0-2.163,0.77-2.163,2.209l-0.008,2.76l 4.959,0 l-0.585,5.514L 18,16L 17.996,32z"></path></g></svg> Share on Facebook</a><a class="btn share" target="_blank" rel="noopener noreferrer" href="https://twitter.com/intent/tweet?url=https://www.ncbi.nlm.nih.gov/books/NBK604953/&text=Multiplex%20Enzyme-Linked%20Immunosorbent%20Assay%20(ELISA)%20for%20Detection%20of%20Human%20Cytokines%20in%20Culture%20Supernatants"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 33 33" style="vertical-align:middle" width="24" height="24"><g><path d="M 32,6.076c-1.177,0.522-2.443,0.875-3.771,1.034c 1.355-0.813, 2.396-2.099, 2.887-3.632 c-1.269,0.752-2.674,1.299-4.169,1.593c-1.198-1.276-2.904-2.073-4.792-2.073c-3.626,0-6.565,2.939-6.565,6.565 c0,0.515, 0.058,1.016, 0.17,1.496c-5.456-0.274-10.294-2.888-13.532-6.86c-0.565,0.97-0.889,2.097-0.889,3.301 c0,2.278, 1.159,4.287, 2.921,5.465c-1.076-0.034-2.088-0.329-2.974-0.821c-0.001,0.027-0.001,0.055-0.001,0.083 c0,3.181, 2.263,5.834, 5.266,6.438c-0.551,0.15-1.131,0.23-1.73,0.23c-0.423,0-0.834-0.041-1.235-0.118 c 0.836,2.608, 3.26,4.506, 6.133,4.559c-2.247,1.761-5.078,2.81-8.154,2.81c-0.53,0-1.052-0.031-1.566-0.092 c 2.905,1.863, 6.356,2.95, 10.064,2.95c 12.076,0, 18.679-10.004, 18.679-18.68c0-0.285-0.006-0.568-0.019-0.849 C 30.007,8.548, 31.12,7.392, 32,6.076z"></path></g></svg> Share on Twitter</a></div></aside><aside id="jr-rtoc-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Table of Content</div></div><div class="cnt lol f1"><a href="/books/n/nciprotocols/?report=reader">Title Information</a><a href="/books/n/nciprotocols/toc/?report=reader">Table of Contents Page</a></div></aside><aside id="jr-help-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Settings</div></div><div class="cnt f1"><div id="jr-typo-p" class="typo"><div><a class="sf btn wsprkl">A-</a><a class="lf btn wsprkl">A+</a></div><div><a class="bcol-auto btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 200 100" preserveAspectRatio="none"><text x="10" y="70" style="font-size:60px;font-family: Trebuchet MS, ArialMT, Arial, sans-serif" textLength="180">AUTO</text></svg></a><a class="bcol-1 btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M15,25 85,25zM15,40 85,40zM15,55 85,55zM15,70 85,70z"></path></svg></a><a class="bcol-2 btn wsprkl"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M5,25 45,25z M55,25 95,25zM5,40 45,40z M55,40 95,40zM5,55 45,55z M55,55 95,55zM5,70 45,70z M55,70 95,70z"></path></svg></a></div></div><div class="lol"><a class="" href="/books/NBK604953/?report=classic">Switch to classic view</a><a href="/books/NBK604953/pdf/Bookshelf_NBK604953.pdf">PDF (675K)</a><a href="/books/NBK604953/?report=printable">Print View</a></div></div></aside><aside id="jr-bkhelp-p" class="hidden flexv"><div class="tb sk-htbar flexh"><div><a class="jr-p-close btn wsprkl">Done</a></div><div class="title-text f1">Help</div></div><div class="cnt f1 lol"><a id="jr-helpobj-sw" data-path="/corehtml/pmc/jatsreader/ptpmc_3.22/" data-href="/corehtml/pmc/jatsreader/ptpmc_3.22/img/bookshelf/help.xml" href="">Help</a><a href="mailto:info@ncbi.nlm.nih.gov?subject=PubReader%20feedback%20%2F%20NBK604953%20%2F%20sid%3ACE8B5AF87C7FFCB1_0191SID%20%2F%20phid%3ACE8CBB6B7C8E176100000000011000FB.4">Send us feedback</a><a id="jr-about-sw" data-path="/corehtml/pmc/jatsreader/ptpmc_3.22/" data-href="/corehtml/pmc/jatsreader/ptpmc_3.22/img/bookshelf/about.xml" href="">About PubReader</a></div></aside><aside id="jr-objectbox" class="thidden hidden"><div class="jr-objectbox-close wsprkl">✘</div><div class="jr-objectbox-inner cnt"><div class="jr-objectbox-drawer"></div></div></aside><nav id="jr-pm-left" class="hidden"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 40 800" preserveAspectRatio="none"><text font-stretch="ultra-condensed" x="800" y="-15" text-anchor="end" transform="rotate(90)" font-size="18" letter-spacing=".1em">Previous Page</text></svg></nav><nav id="jr-pm-right" class="hidden"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 40 800" preserveAspectRatio="none"><text font-stretch="ultra-condensed" x="800" y="-15" text-anchor="end" transform="rotate(90)" font-size="18" letter-spacing=".1em">Next Page</text></svg></nav><nav id="jr-fip" class="hidden"><nav id="jr-fip-term-p"><input type="search" placeholder="search this page" id="jr-fip-term" autocorrect="off" autocomplete="off" /><a id="jr-fip-mg" class="wsprkl btn" title="Find"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 550 600" preserveAspectRatio="none"><path fill="none" stroke="#000" stroke-width="36" stroke-linecap="round" style="fill:#FFF" d="m320,350a153,153 0 1,0-2,2l170,170m-91-117 110,110-26,26-110-110"></path></svg></a><a id="jr-fip-done" class="wsprkl btn" title="Dismiss find">✘</a></nav><nav id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK604953_"><span class="label">NCL Method ITA-27</span><span class="title" itemprop="name">Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants</span></h1><div class="subtitle whole_rhythm">Version 1</div><p class="contribs">Cedrone E, Potter TM, Neun BW, et al.</p><p class="fm-aai"><a href="#_NBK604953_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="ncipr64.s1"><h2 id="_ncipr64_s1_">1. Introduction</h2><p>Cytokines, chemokines and interferons (IFN) are soluble mediators of inflammation and a variety of responses that orchestrate both innate and adaptive immunity in health and disease [<a class="bibr" href="#ncipr64.ref1" rid="ncipr64.ref1">1</a>–<a class="bibr" href="#ncipr64.ref10" rid="ncipr64.ref10">10</a>]. Understanding the induction of these biomarkers in response to a drug product helps to establish the product’s safety profile and shed light on the mechanism of action or mechanism of toxicity. This document describes experimental procedure for analysis of culture supernatants by multiplex ELISA to detect presence of type I IFNs (IFNα, IFNβ, IFNω), type II IFN (IFNγ), type III IFN (IFNλ), cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-21, IL-22, IL-23, IL-27, IP-10, MCP-1, MCP-2, MIP-1α, MIP-1β, RANTES, TNFα) in culture supernatants. NCL protocol ITA-10 should be referred to for details of preparation of culture supernatants. Protocols for single-plex ELISA (ITA-22, ITA-23, ITA-24 and ITA-25) can also be considered. The advantage of this protocol over single-plex ELISA is that it allows simultaneous analysis of a broad spectrum of markers.</p></div><div id="ncipr64.s2"><h2 id="_ncipr64_s2_">2. Principle</h2><p>A 96-well plate is coated with capture antibodies specific to the target cytokines and immobilized at particular location within each well. Cell culture supernatants are loaded onto the plate and cytokines present in the supernatant are captured by the antibodies. The excess sample is washed away, and captured cytokines are detected with secondary antibody conjugated to biotin. Streptavidin-conjugated horse radish peroxidase is then used to detect captured analytes. After additional wash, the conjugate remaining at each location is detected using chemiluminescent substrate. Images are taken using Quansys Image Pro reader and analyzed using Q-View Software. The intensity of the individual spot corresponding to a particular analyte is proportional to the level of the analyte in the test supernatant. The quantities of the individual cytokines are determined by comparing the intensity of spots corresponding to each of the cytokines in the test sample to that in the standard curve comprised of various concentrations of reference standards. The ELISA procedure described herein takes approximately 6 hours to complete. If ELISA cannot be conducted immediately after incubation of whole blood/PBMC with nanoparticles is complete, the culture supernatants can be stored at −80°C.</p></div><div id="ncipr64.s3"><h2 id="_ncipr64_s3_">3. Reagents, Materials, and Equipment</h2><blockquote><p><i>Note: The NCL does not endorse any of the suppliers listed below; these reagents were used in the development of the protocol and their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted. Please note that suppliers may undergo a name change due to a variety of factors. Brands and part numbers typically remain consistent but may also change over time</i>.</p></blockquote><dl id="ncipr64.l1" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr64.lt1"><p class="no_top_margin">Reagents
|
|
<dl id="ncipr64.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.1.</dt><dd id="ncipr64.lt2"><p class="no_top_margin">Custom Multiplex kit, (Quansys, 107749GR-Q-Plex<sup>™</sup> Custom Chemi Kit)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.2.</dt><dd id="ncipr64.lt3"><p class="no_top_margin">Phosphate buffered saline (PBS), (GE Life Science, SH30256.01)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.3.</dt><dd id="ncipr64.lt4"><p class="no_top_margin">Cell culture grade water, GE Life Science, SH30529.02)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.4.</dt><dd id="ncipr64.lt5"><p class="no_top_margin">RPMI1640, (GE Life Sciences, Hyclone, SH30096.01)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.5.</dt><dd id="ncipr64.lt6"><p class="no_top_margin">Pen/Strep solution, (GE Life Sciences, Hyclone, SV30010)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.6.</dt><dd id="ncipr64.lt7"><p class="no_top_margin">Cytokine Calibration Standards (see <a href="#ncipr64.s10">section 10</a> for the example of Product Information Card showing individual cytokine’s concentrations)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr64.lt8"><p class="no_top_margin">Materials
|
|
<dl id="ncipr64.l3" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.2.1.</dt><dd id="ncipr64.lt9"><p class="no_top_margin">96-well, U-bottom plates</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.2.</dt><dd id="ncipr64.lt10"><p class="no_top_margin">96-well, V-bottom polypropylene plates</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.3.</dt><dd id="ncipr64.lt11"><p class="no_top_margin">Pipettes covering the range from 0.05 to 1 mL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.4.</dt><dd id="ncipr64.lt12"><p class="no_top_margin">Microcentrifuge tubes, 1.5 mL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.5.</dt><dd id="ncipr64.lt13"><p class="no_top_margin">Multichannel pipette (8 or 12-channels)</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.6.</dt><dd id="ncipr64.lt14"><p class="no_top_margin">Reagent reservoirs</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr64.lt15"><p class="no_top_margin">Equipment
|
|
<dl id="ncipr64.l4" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.3.1.</dt><dd id="ncipr64.lt16"><p class="no_top_margin">Microcentrifuge</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.2.</dt><dd id="ncipr64.lt17"><p class="no_top_margin">Refrigerator, 2-8°C</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.3.</dt><dd id="ncipr64.lt18"><p class="no_top_margin">Freezer, −80°C</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.4.</dt><dd id="ncipr64.lt19"><p class="no_top_margin">Vortex</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.5.</dt><dd id="ncipr64.lt20"><p class="no_top_margin">Plate shaker</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.6.</dt><dd id="ncipr64.lt21"><p class="no_top_margin">Plate washer</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.7.</dt><dd id="ncipr64.lt22"><p class="no_top_margin">Multiplex ELISA plate reader capable of reading chemiluminescence</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr64.s4"><h2 id="_ncipr64_s4_">4. Preparation of Reagents and Controls</h2><dl id="ncipr64.l5" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr64.lt23"><p class="no_top_margin">Calibrators</p><p>The kits come with 1 to 3 vials of lyophilized calibrators depending on the format being used. Each set of calibrators should be prepared with <b>sample diluent</b> according to the product information card included with the kit (see <a href="#ncipr64.s10">section 10</a>). The calibrators will then be serially diluted, 3-fold, as described in the procedure below (see <a href="#ncipr64.lt34">step 6.3</a>).</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.</dt><dd id="ncipr64.lt24"><p class="no_top_margin">Sample diluent (10 mL): Use as is.</p></dd></dl><dl class="bkr_refwrap"><dt>4.3.</dt><dd id="ncipr64.lt25"><p class="no_top_margin">Assay diluent (lyophilized): Reconstitute with 6 mL Cell Culture Grade water.</p></dd></dl><dl class="bkr_refwrap"><dt>4.4.</dt><dd id="ncipr64.lt26"><p class="no_top_margin">Detection reagent (lyophilized): Reconstitute with 6 mL Cell Culture Grade water.</p></dd></dl><dl class="bkr_refwrap"><dt>4.5.</dt><dd id="ncipr64.lt27"><p class="no_top_margin">Streptavidin-HRP (6 mL): Use as is.</p></dd></dl><dl class="bkr_refwrap"><dt>4.6.</dt><dd id="ncipr64.lt28"><p class="no_top_margin">Substrate A (3 mL) and Substrate B (3 mL): Mix both reagents together 15 minutes before use and store in the dark at room temperature.</p></dd></dl><dl class="bkr_refwrap"><dt>4.7.</dt><dd id="ncipr64.lt29"><p class="no_top_margin">Wash buffer, 20X (50 mL): Mix with 950 mL of distilled water for a 1X solution.</p></dd></dl></dl></div><div id="ncipr64.s5"><h2 id="_ncipr64_s5_">5. Preparation of Inhibition/Enhancement Controls (IECs)</h2><p>Two approaches can be used to prepare IECs:
|
|
<dl id="ncipr64.l6" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.</dt><dd id="ncipr64.lt30"><p class="no_top_margin"><u>Approach A</u></p><p>Use culture supernatant from the positive control sample and spike it with the test nanoparticle at 4 concentrations (refer to NCL ITA-10). For example, add 6 μL of 10X nanoparticle working solution into 60 μL of positive control supernatant. The final concentration of nanoparticle in this sample will mimic that in the supernatants from nanoparticle treated cells. The concentration of the analyte in the IEC supernatant will be 1.1X lower. Compare the analyte level in the positive control supernatant with that in IEC x 1.1 to account for the dilution factor. If the difference in test results is within 25%, testnanoparticle does not interfere with ELISA.</p></dd></dl><dl class="bkr_refwrap"><dt>5.2.</dt><dd id="ncipr64.lt31"><p class="no_top_margin"><u>Approach B</u></p><p>Use cell free controls and spike them with calibration standard. For example, add 6 μL of calibrator 1 from <a href="#ncipr64.lt34">step 6.3</a> to 60 μL of cell-free supernatant from ITA-10. This IEC should be approximately comparable to calibrator 3 (<a href="#ncipr64.lt37">step 6.3.3</a>). If the difference in test results is within 25%, test-nanoparticle does not interfere with ELISA.</p></dd></dl></dl></p></div><div id="ncipr64.s6"><h2 id="_ncipr64_s6_">6. Experimental Procedure for 4-plex Interferon Multiplex</h2><dl id="ncipr64.l7" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>6.1.</dt><dd id="ncipr64.lt32"><p class="no_top_margin">Prepare assay diluent, sample diluent and wash buffer as described in <a href="#ncipr64.s4">Section 4</a>. Store all buffers at room temperature and use on the same day as preparation.</p></dd></dl><dl class="bkr_refwrap"><dt>6.2.</dt><dd id="ncipr64.lt33"><p class="no_top_margin">Reconstitute calibrator(s) with sample diluent according to the included information card (see <a href="#ncipr64.s10">Section 10</a> for an example information card).</p></dd></dl><dl class="bkr_refwrap"><dt>6.3.</dt><dd id="ncipr64.lt34"><p class="no_top_margin">Prepare calibration standards in microtubes or a 96-well tray as follows:
|
|
<dl id="ncipr64.l8" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>6.3.1.</dt><dd id="ncipr64.lt35"><p class="no_top_margin">Add 200 μL of the stock calibrator to the first tube/well labeled Standard or Calibrator 1.</p></dd></dl><dl class="bkr_refwrap"><dt>6.3.2.</dt><dd id="ncipr64.lt36"><p class="no_top_margin">Add 120 μL of the <b>sample diluent</b> to an additional 7 tubes/wells labeled sequentially as Standard or Calibrator 2 through 8.</p></dd></dl><dl class="bkr_refwrap"><dt>6.3.3.</dt><dd id="ncipr64.lt37"><p class="no_top_margin">Perform a serial, 3-fold dilution of the first standard by transferring 60 μL of the Standard 1 to the Standard 2 tube/well, mix by repeated up-and-down pipetting, continue transferring 60 μL serially into the next tubes/wells until Standard 7 is reached. Standard 8 is sample diluent only (see image below).</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>6.4.</dt><dd id="ncipr64.lt38"><p class="no_top_margin">Thaw frozen culture supernatants at room temperature or use freshly collected supernatants.</p></dd></dl><dl class="bkr_refwrap"><dt>6.5.</dt><dd id="ncipr64.lt39"><p class="no_top_margin">Dilute culture supernatants 2-fold with <b>sample diluent</b> by mixing 60 μL of the culture supernatants with 60 μL of the <b>sample diluent</b>.</p></dd></dl><dl class="bkr_refwrap"><dt>6.6.</dt><dd id="ncipr64.lt40"><p class="no_top_margin">Add 50 μL of the <b>assay diluent</b> to each well on the multiplex ELISA plate.</p></dd></dl><dl class="bkr_refwrap"><dt>6.7.</dt><dd id="ncipr64.lt41"><p class="no_top_margin">After adding the <b>assay diluent</b>, load 50 μL of the standards (<a href="#ncipr64.lt34">step 6.3</a>) and culture supernatants (<a href="#ncipr64.lt39">step 6.5</a>) to the 96-well multiplex plate, seal and incubate at room temperature on a shaker set at approximately 500 rpm for 2 hours.</p></dd></dl><dl class="bkr_refwrap"><dt>6.8.</dt><dd id="ncipr64.lt42"><p class="no_top_margin">Wash the plate 3 times with the wash buffer prepared in <a href="#ncipr64.lt32">step 6.1</a> (<a href="#ncipr64.lt29">section 4.7</a>). Use 300 μL of the wash buffer per well. Tap the plate on paper towel to remove excess buffer and immediately proceed to the next step.</p></dd></dl><dl class="bkr_refwrap"><dt>6.9.</dt><dd id="ncipr64.lt43"><p class="no_top_margin">Add 50 μL per well of previously prepared detection reagent (<a href="#ncipr64.lt26">section 4.4</a>), seal and incubate the plate at room temperature on a shaker set to approximately 500 rpm for 1 hour.</p></dd></dl><dl class="bkr_refwrap"><dt>6.10.</dt><dd id="ncipr64.lt44"><p class="no_top_margin">Repeat <a href="#ncipr64.lt42">step 6.8</a>.</p></dd></dl><dl class="bkr_refwrap"><dt>6.11.</dt><dd id="ncipr64.lt45"><p class="no_top_margin">Add 50 μL per well of the ready-to-use Streptavidin-HRP conjugate provided with the kit, seal and incubate the plate at room temperature on a shaker set to approximately 500 rpm for 15 minutes.</p></dd></dl><dl class="bkr_refwrap"><dt>6.12.</dt><dd id="ncipr64.lt46"><p class="no_top_margin">Repeat <a href="#ncipr64.lt42">step 6.8</a> two times and immediately proceed to the next step.</p></dd></dl><dl class="bkr_refwrap"><dt>6.13.</dt><dd id="ncipr64.lt47"><p class="no_top_margin">Add 50 μL per well of the previously prepared ChemiLum substrate (<a href="#ncipr64.lt28">section 4.6</a>).</p></dd></dl><dl class="bkr_refwrap"><dt>6.14.</dt><dd id="ncipr64.lt48"><p class="no_top_margin">Read the plate using the Quansys ImagePro reader.</p></dd></dl></dl><div id="ncipr64.fig1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ncipr64f1&p=BOOKS&id=604953_ncipr64f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK604953/bin/ncipr64f1.jpg" alt="test tubes showing serial dilution" class="tileshop" title="Click on image to zoom" /></a></div></div></div><div id="ncipr64.s7"><h2 id="_ncipr64_s7_">7. Experimental Procedure for 14-plex and 15-plex multiplex</h2><p>The procedure for both the 14-plex and 15-plex is the same as the IFN 4-plex, with the exception that in <b><u>the 15-plex there is no assay buffer</u></b> as in <a href="#ncipr64.lt40">step 6.6</a>.</p></div><div id="ncipr64.s8"><h2 id="_ncipr64_s8_">8. Acceptance Criteria</h2><dl id="ncipr64.l9" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>8.1.</dt><dd id="ncipr64.lt49"><p class="no_top_margin">The % CV and PDFT for each calibration standard and quality control should be within 25%.</p></dd></dl><dl class="bkr_refwrap"><dt>8.2.</dt><dd id="ncipr64.lt50"><p class="no_top_margin">The % CV for each test sample, including supernatants from whole blood cultures treated with positive control, negative control and nanoparticle samples, should be within 25%. At least one replicate of positive and negative control should be acceptable for run to be accepted.</p></dd></dl><dl class="bkr_refwrap"><dt>8.3.</dt><dd id="ncipr64.lt51"><p class="no_top_margin">If both replicates of positive control or negative control fail to meet acceptance criterion described in 8.2 the run should be repeated.</p></dd></dl><dl class="bkr_refwrap"><dt>8.4.</dt><dd id="ncipr64.lt52"><p class="no_top_margin">Within the acceptable run, if two of three replicates of unknown sample fail to meet acceptance criterion described in 8.2, this unknown sample should be re-analyzed.</p></dd></dl></dl></div><div id="ncipr64.s9"><h2 id="_ncipr64_s9_">9. Example of ELISA Plate Template</h2><div id="ncipr64.fig2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Rows%20G%20%26%20H%20do%20not%20contain%20cells&p=BOOKS&id=604953_ncipr64f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK604953/bin/ncipr64f2.jpg" alt="Rows G & H do not contain cells" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Rows G & H do not contain cells.</p><p>Std: standard; B0 = blank (assay diluent); Unt: Untreated supernatant; NC: negative control supernatant; PC: positive control supernatant; VC: vehicle control supernatant; TS1-4: test sample supernatants at concentrations 1-4; IEC1-4: inhibition enhancement controls for TS1-4 concentrations; CF: cell free</p></div></div></div><div id="ncipr64.s10"><h2 id="_ncipr64_s10_">10. Example of Product Information Card</h2><div id="ncipr64.fig3" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ncipr64f3&p=BOOKS&id=604953_ncipr64f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK604953/bin/ncipr64f3.jpg" alt="Picture of a product information card. Provides information such as analyte, concentration, product codes, lot numbers." class="tileshop" title="Click on image to zoom" /></a></div></div></div><div id="ncipr64.rl.r1"><h2 id="_ncipr64_rl_r1_">11. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ncipr64.ref1">Bioanalytical Chemistry of Cytokines-A Review. Julie
|
|
A. Stenken, Andreas
|
|
J. Poschenrieder. Anal Chim Acta. Anal Chim Acta. 2015
|
|
Jan
|
|
1; 853: 95–115. doi: 10.1016/j.aca.2014.10.009
|
|
PMCID: PMC4717841 [<a href="/pmc/articles/PMC4717841/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4717841</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25467452" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25467452</span></a>] [<a href="http://dx.crossref.org/10.1016/j.aca.2014.10.009" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ncipr64.ref2">IL-1 and related cytokines in innate and adaptive immunity in health and disease. Alberto
|
|
Mantovani, Charles A.
|
|
Dinarello, Martina
|
|
Molgora, Cecilia
|
|
Garlanda. Immunity. 2019
|
|
Apr
|
|
16; 50(4): 778–795. doi: 10.1016/j.immuni.2019.03.012
|
|
PMCID: PMC7174020
|
|
[<a href="/pmc/articles/PMC7174020/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC7174020</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/30995499" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30995499</span></a>] [<a href="http://dx.crossref.org/10.1016/j.immuni.2019.03.012" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ncipr64.ref3">Th1 cytokines, true functional signatures for protective immunity against TB?
|
|
Gucheng
|
|
Zeng, Guoliang
|
|
Zhang, Xinchun
|
|
Chen. Cell Mol Immunol. 2018
|
|
Mar; 15(3): 206–215. doi: 10.1038/cmi.2017.113
|
|
PMCID: PMC5843617
|
|
[<a href="/pmc/articles/PMC5843617/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5843617</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/29151578" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 29151578</span></a>] [<a href="http://dx.crossref.org/10.1038/cmi.2017.113" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ncipr64.ref4">Inflammation and Pancreatic Cancer: Focus on Metabolism, Cytokines, and Immunity. Andrea
|
|
Padoan, Mario
|
|
Plebani, Daniela
|
|
Basso. Int J Mol Sci. 2019
|
|
Feb; 20(3): 676. doi: 10.3390/ijms20030676
|
|
PMCID: PMC6387440
|
|
[<a href="/pmc/articles/PMC6387440/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6387440</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/30764482" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30764482</span></a>] [<a href="http://dx.crossref.org/10.3390/ijms20030676" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ncipr64.ref5">Innate cell communication kick-starts pathogen-specific immunity. Amariliz
|
|
Rivera, Mark C.
|
|
Siracusa, George S.
|
|
Yap, William C.
|
|
Gause. Nat Immunol. 2016
|
|
Mar
|
|
22; 17(4): 356–363. doi: 10.1038/ni.3375
|
|
PMCID: PMC4949486
|
|
[<a href="/pmc/articles/PMC4949486/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4949486</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/27002843" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 27002843</span></a>] [<a href="http://dx.crossref.org/10.1038/ni.3375" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ncipr64.ref6">Cytokines: Names and Numbers You Should Care About. Stephen R.
|
|
Holdsworth, Poh-Yi
|
|
Gan. Clin J Am Soc Nephrol. 2015
|
|
Dec
|
|
7; 10(12): 2243–2254. doi: 10.2215/CJN.07590714
|
|
PMCID: PMC4670773
|
|
[<a href="/pmc/articles/PMC4670773/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4670773</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/25941193" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25941193</span></a>] [<a href="http://dx.crossref.org/10.2215/CJN.07590714" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ncipr64.ref7">The immunobiology of the Interleukin-12 family: Room for discovery. Elia D.
|
|
Tait Wojno, Christopher A.
|
|
Hunter, Jason S.
|
|
Stumhofer. Immunity. 2019
|
|
Apr
|
|
16; 50(4): 851–870. doi: 10.1016/j.immuni.2019.03.011
|
|
PMCID: PMC6472917
|
|
[<a href="/pmc/articles/PMC6472917/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6472917</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/30995503" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30995503</span></a>] [<a href="http://dx.crossref.org/10.1016/j.immuni.2019.03.011" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ncipr64.ref8">Innate control of adaptive immunity: Beyond the three-signal paradigm. Aakanksha
|
|
Jain, Chandrashekhar
|
|
Pasare. J Immunol. 2017
|
|
May
|
|
15; 198(10): 3791–3800. doi: 10.4049/jimmunol.1602000
|
|
PMCID: PMC5442885
|
|
[<a href="/pmc/articles/PMC5442885/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5442885</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/28483987" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 28483987</span></a>] [<a href="http://dx.crossref.org/10.4049/jimmunol.1602000" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ncipr64.ref9">Cytokines and Long Noncoding RNAs. Susan
|
|
Carpenter, Katherine A.
|
|
Fitzgerald. Cold Spring Harb Perspect Biol. 2018
|
|
Jun; 10(6): a028589. doi: 10.1101/cshperspect.a028589
|
|
PMCID: PMC5983188
|
|
[<a href="/pmc/articles/PMC5983188/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5983188</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/28716885" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 28716885</span></a>] [<a href="http://dx.crossref.org/10.1101/cshperspect.a028589" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ncipr64.ref10">Th17 cytokines in mucosal immunity and inflammation. Lokesh
|
|
Guglani, Shabaana A.
|
|
Khader. Curr Opin HIV AIDS. 2010
|
|
Mar; 5(2): 120–127. doi: 10.1097/COH.0b013e328335c2f6
|
|
PMCID: PMC2892849
|
|
[<a href="/pmc/articles/PMC2892849/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2892849</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20543588" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20543588</span></a>] [<a href="http://dx.crossref.org/10.1097/COH.0b013e328335c2f6" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></dl></div><div id="ncipr64.s11"><h2 id="_ncipr64_s11_">12. Abbreviations</h2><dl><dt id="ncipr64.abb_DL1_DI1">B0</dt><dd><p>blank (assay diluent)</p></dd><dt id="ncipr64.abb_DL1_DI2">CF</dt><dd><p>cell free</p></dd><dt id="ncipr64.abb_DL1_DI3">IEC</dt><dd><p>inhibition enhancement control</p></dd><dt id="ncipr64.abb_DL1_DI4">IFN</dt><dd><p>interferon</p></dd><dt id="ncipr64.abb_DL1_DI5">LLD</dt><dd><p>lower limit of detection</p></dd><dt id="ncipr64.abb_DL1_DI6">LPS</dt><dd><p>lipopolysaccharide</p></dd><dt id="ncipr64.abb_DL1_DI7">NC</dt><dd><p>negative control supernatant</p></dd><dt id="ncipr64.abb_DL1_DI8">PBMC</dt><dd><p>peripheral blood mononuclear cells</p></dd><dt id="ncipr64.abb_DL1_DI9">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr64.abb_DL1_DI10">PC</dt><dd><p>positive control supernatant</p></dd><dt id="ncipr64.abb_DL1_DI11">PHA-M</dt><dd><p>phytohemagglutinin-M</p></dd><dt id="ncipr64.abb_DL1_DI12">Std</dt><dd><p>standard</p></dd><dt id="ncipr64.abb_DL1_DI13">TS</dt><dd><p>test sample supernatant</p></dd><dt id="ncipr64.abb_DL1_DI14">ULD</dt><dd><p>upper limit of detection</p></dd><dt id="ncipr64.abb_DL1_DI15">VC</dt><dd><p>vehicle control supernatant</p></dd></dl></div><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_top_margin"><p>This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.</p><p>The method was developed in collaboration between Nanotechnology Characterization Lab and Quansys Biosciences.</p></p></div></dd></dl><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ncipr64.fn1"><p class="no_top_margin">address correspondence to: <a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></p></div></dd></dl><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_top_margin"><div>
|
|
<span class="mixed-citation" id="ncipr64.suggestedcitation">Cedrone E, Potter TM, Neun BW, Tyler A, Dobrovolskaia MA, NCL Method ITA-27: Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants. <a href="https://ncl.cancer.gov/resources/assay-cascade-protocols" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://ncl.cancer.gov/resources/assay-cascade-protocols</a> DOI: <a href="http://dx.crossref.org/10.17917/P2T3-8P84" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/P2T3-8P84</a></span>
|
|
</div></p></div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK604953_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Edward Cedrone</span>,<sup>1</sup> <span itemprop="author">Timothy M. Potter</span>,<sup>1</sup> <span itemprop="author">Barry W. Neun</span>,<sup>1</sup> <span itemprop="author">Abby Tyler</span>,<sup>2</sup> and <span itemprop="author">Marina A. Dobrovolskaia</span><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>1</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup> Nanotechnology Characterization Lab, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, MD 21702<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@aniram" class="oemail">vog.hin.liam@aniram</a></div></div><div class="affiliation"><sup>2</sup> Quansys Biosciences, Logan, UT</div><div><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author.</div><h3>Publication History</h3><p class="small">Published: <span itemprop="datePublished">September 2020</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p><a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Cancer Institute (US)</a>, Bethesda (MD)</p><h3>NLM Citation</h3><p>Cedrone E, Potter TM, Neun BW, et al. Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Human Cytokines in Culture Supernatants: Version 1. 2020 Sep. In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method ITA-27.<span class="bk_cite_avail"></span> doi: 10.17917/P2T3-8P84</p></div><div class="small-screen-prev"><a href="/books/n/nciprotocols/ncipr65/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/nciprotocols/ncipr62/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobncipr64fig1"><div id="ncipr64.fig1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ncipr64f1&p=BOOKS&id=604953_ncipr64f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK604953/bin/ncipr64f1.jpg" alt="test tubes showing serial dilution" class="tileshop" title="Click on image to zoom" /></a></div></div></article><article data-type="fig" id="figobncipr64fig2"><div id="ncipr64.fig2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Rows%20G%20%26%20H%20do%20not%20contain%20cells&p=BOOKS&id=604953_ncipr64f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK604953/bin/ncipr64f2.jpg" alt="Rows G & H do not contain cells" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>Rows G & H do not contain cells.</p><p>Std: standard; B0 = blank (assay diluent); Unt: Untreated supernatant; NC: negative control supernatant; PC: positive control supernatant; VC: vehicle control supernatant; TS1-4: test sample supernatants at concentrations 1-4; IEC1-4: inhibition enhancement controls for TS1-4 concentrations; CF: cell free</p></div></div></article><article data-type="fig" id="figobncipr64fig3"><div id="ncipr64.fig3" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ncipr64f3&p=BOOKS&id=604953_ncipr64f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK604953/bin/ncipr64f3.jpg" alt="Picture of a product information card. Provides information such as analyte, concentration, product codes, lot numbers." class="tileshop" title="Click on image to zoom" /></a></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
|
|
|
|
|
|
|
|
|
|
<!-- Book content -->
|
|
|
|
<script type="text/javascript" src="/portal/portal3rc.fcgi/rlib/js/InstrumentNCBIBaseJS/InstrumentPageStarterJS.js"> </script>
|
|
|
|
|
|
<!-- CE8B5AF87C7FFCB1_0191SID /projects/books/PBooks@9.11 portal105 v4.1.r689238 Tue, Oct 22 2024 16:10:51 -->
|
|
<span id="portal-csrf-token" style="display:none" data-token="CE8B5AF87C7FFCB1_0191SID"></span>
|
|
|
|
<script type="text/javascript" src="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/js/3968615.js" snapshot="books"></script></body>
|
|
</html>
|