150 lines
58 KiB
Text
150 lines
58 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="NBK604912">
|
|
<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="/NBK604912/?report=reader">
|
|
<meta name="ncbi_pagename" content="Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA - 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>Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA - 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/ncipr44/?report=reader">
|
|
<meta name="jr-next-unit" content="/books/n/nciprotocols/ncipr39/?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="Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA">
|
|
<meta name="citation_publisher" content="National Cancer Institute (US)">
|
|
<meta name="citation_date" content="2020/05">
|
|
<meta name="citation_author" content="Barry W. Neun">
|
|
<meta name="citation_author" content="Edward Cedrone">
|
|
<meta name="citation_author" content="Marina A. Dobrovolskaia">
|
|
<meta name="citation_pmid" content="39013064">
|
|
<meta name="citation_doi" content="10.17917/EQCT-3C51">
|
|
<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK604912/">
|
|
<link rel="schema.DC" href="http://purl.org/DC/elements/1.0/">
|
|
<meta name="DC.Title" content="Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA">
|
|
<meta name="DC.Type" content="Text">
|
|
<meta name="DC.Publisher" content="National Cancer Institute (US)">
|
|
<meta name="DC.Contributor" content="Barry W. Neun">
|
|
<meta name="DC.Contributor" content="Edward Cedrone">
|
|
<meta name="DC.Contributor" content="Marina A. Dobrovolskaia">
|
|
<meta name="DC.Date" content="2020/05">
|
|
<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK604912/">
|
|
<meta name="description" content="This document describes a protocol for quantitative determination of complement activation by an Enzyme Immunoassay (EIA). The complement system represents an innate arm of immune defense and is named so because it “complements” the antibody-mediated immune response. Three major pathways leading to complement activation have been described: they are the classical pathway, alternative pathway and lectin pathway (Figure 1). The classical pathway is activated by immune (antigen-antibody) complexes. Activation of the alternative pathway is antibody independent. The lectin pathway is initiated by plasma protein mannose-binding lectin.">
|
|
<meta name="og:title" content="Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA">
|
|
<meta name="og:type" content="book">
|
|
<meta name="og:description" content="This document describes a protocol for quantitative determination of complement activation by an Enzyme Immunoassay (EIA). The complement system represents an innate arm of immune defense and is named so because it “complements” the antibody-mediated immune response. Three major pathways leading to complement activation have been described: they are the classical pathway, alternative pathway and lectin pathway (Figure 1). The classical pathway is activated by immune (antigen-antibody) complexes. Activation of the alternative pathway is antibody independent. The lectin pathway is initiated by plasma protein mannose-binding lectin.">
|
|
<meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK604912/">
|
|
<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/ncipr42/?report=reader">
|
|
<link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK604912/">
|
|
<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="CE8E29D17C8E7D710000000000CB00B4.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/NBK604912/?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/ncipr44/?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-5.2, Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA: Version 3</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/ncipr39/?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/NBK604912/"><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/NBK604912/&text=Analysis%20of%20Complement%20Activation%20by%20Single-Plex%20EIA%20or%20Multiplex%20ELISA"><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/NBK604912/?report=classic">Switch to classic view</a><a href="/books/NBK604912/pdf/Bookshelf_NBK604912.pdf">PDF (550K)</a><a href="/books/NBK604912/?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%20NBK604912%20%2F%20sid%3ACE8B5AF87C7FFCB1_0191SID%20%2F%20phid%3ACE8E29D17C8E7D710000000000CB00B4.4">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="_NBK604912_"><span class="label">NCL Method ITA-5.2</span><span class="title" itemprop="name">Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA</span></h1><div class="subtitle whole_rhythm">Version 3</div><p class="contribs">Neun BW, Cedrone E, Dobrovolskaia MA.</p><p class="fm-aai"><a href="#_NBK604912_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="ncipr42.s1"><h2 id="_ncipr42_s1_">1. Introduction</h2><p>This document describes a protocol for quantitative determination of complement activation by an Enzyme Immunoassay (EIA). The complement system represents an innate arm of immune defense and is named so because it “complements” the antibody-mediated immune response. Three major pathways leading to complement activation have been described: they are the classical pathway, alternative pathway and lectin pathway (<a class="figpopup" href="/books/NBK604912/figure/ncipr42.fig1/?report=objectonly" target="object" rid-figpopup="figncipr42fig1" rid-ob="figobncipr42fig1">Figure 1</a>). The classical pathway is activated by immune (antigen-antibody) complexes. Activation of the alternative pathway is antibody independent. The lectin pathway is initiated by plasma protein mannose-binding lectin.</p><p>The complement system is a group of ~30 protein that includes several components (C1 - C9), and Factors (B, D, H, I, and P). Activation of any of the three pathways results in cleavage of the C3 component of the complement system [<a class="bibr" href="#ncipr42.ref1" rid="ncipr42.ref1">1</a>, <a class="bibr" href="#ncipr42.ref2" rid="ncipr42.ref2">2</a>].</p><p>This protocol is intended for follow-up studies on samples which demonstrated a positive response in the qualitative assay (NCL Method ITA 5.1). It can also be performed as an independent protocol when high throughput analysis is needed.</p></div><div id="ncipr42.s2"><h2 id="_ncipr42_s2_">2. Principles</h2><p>In the protocol presented herein, human plasma is exposed to a test material and subsequently analyzed by EIA for the presence of the complement components C4d, iC3b and Bb. The antibodies specific to these proteins are immobilized on 96 well plates and are obtained from commercial suppliers. Test nanoparticles found to be positive in the qualitative western blot assay are then subject to a more detailed investigation aimed at delineation of the specific complement activation pathway. Detection of elevated levels of C4d protein is indicative of complement activation via the classical or lectin pathway. Elevation in Bb levels is a sign of alternative pathway activation. Estimation of iC3b levels is used to confirm, in a more accurate, quantitative way, the results of the initial western blot screen specific to the C3 component of the complement system.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figncipr42fig1" co-legend-rid="figlgndncipr42fig1"><a href="/books/NBK604912/figure/ncipr42.fig1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figncipr42fig1" rid-ob="figobncipr42fig1"><img class="small-thumb" src="/books/NBK604912/bin/ncipr42f1.gif" src-large="/books/NBK604912/bin/ncipr42f1.jpg" alt="Figure 1. Complement activation pathways." /></a><div class="icnblk_cntnt" id="figlgndncipr42fig1"><h4 id="ncipr42.fig1"><a href="/books/NBK604912/figure/ncipr42.fig1/?report=objectonly" target="object" rid-ob="figobncipr42fig1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Complement activation pathways. (This illustration is reproduced from reference 1 with permission from EMD Biosciences, Inc.) </p></div></div></div><div id="ncipr42.s3"><h2 id="_ncipr42_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="ncipr42.l1" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr42.lt1"><p class="no_top_margin">Reagents
|
|
<dl id="ncipr42.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.1.</dt><dd id="ncipr42.lt2"><p class="no_top_margin">Sterile Ca<sup>2+</sup>/Mg<sup>2+</sup>-free phosphate buffered saline (PBS) (GE Life Sciences, SH30256.01)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.2.</dt><dd id="ncipr42.lt3"><p class="no_top_margin">Cobra Venom Factor (positive control) (Quidel Corp., A600)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.3.</dt><dd id="ncipr42.lt4"><p class="no_top_margin">Veronal Buffer (Boston BioProducts, IBB-260)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.4.</dt><dd id="ncipr42.lt5"><p class="no_top_margin">Pooled human plasma, anti-coagulated with Sodium citrate</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.5.</dt><dd id="ncipr42.lt6"><p class="no_top_margin">MicroVue iC3b EIA kit (Quidel Corp., A006)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.6.</dt><dd id="ncipr42.lt7"><p class="no_top_margin">MicroVue C4d fragment EIA kit (Quidel Corp., A0008)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.7.</dt><dd id="ncipr42.lt8"><p class="no_top_margin">MicroVue Bb Plus EIA kit (Quidel Corp., A027)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.8.</dt><dd id="ncipr42.lt9"><p class="no_top_margin">MicroVue Complement Multiplex (8-plex) EIA Kit (Quidel Corp., A900)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.9.</dt><dd id="ncipr42.lt10"><p class="no_top_margin">Doxil (Doxorubicin HCl, liposome, injection) <i>This is a prescription medication available from a licensed pharmacy and may not be available to some research laboratories</i>.</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.10.</dt><dd id="ncipr42.lt11"><p class="no_top_margin">Cremophor, (Sigma, C 5135)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.11.</dt><dd id="ncipr42.lt12"><p class="no_top_margin">Complement activator (Quidel, <a href="https://www.quidel.com/research/complement-reagents/complement-activator" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://www<wbr style="display:inline-block"></wbr>​.quidel.com<wbr style="display:inline-block"></wbr>​/research/complement-reagents<wbr style="display:inline-block"></wbr>​/complement-activator</a>)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.12.</dt><dd id="ncipr42.lt13"><p class="no_top_margin">Taxol (Paclitaxel in Cremophor EL) <i>This is a prescription medication available from a licensed pharmacy and may not be available to some research laboratories</i>.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr42.lt14"><p class="no_top_margin">Materials
|
|
<dl id="ncipr42.l3" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.2.1.</dt><dd id="ncipr42.lt15"><p class="no_top_margin">Pipettes covering the range from 0.05 to 1 mL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.2.</dt><dd id="ncipr42.lt16"><p class="no_top_margin">Microcentrifuge tubes, 1.5 mL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.3.</dt><dd id="ncipr42.lt17"><p class="no_top_margin">Pipet tips, 0.5 µL – 1.0 mL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.4.</dt><dd id="ncipr42.lt18"><p class="no_top_margin">Multichannel (8-12 channel) pipettor with volumes 50-300 µL</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.5.</dt><dd id="ncipr42.lt19"><p class="no_top_margin">15 and 50 mL conical tubes</p></dd></dl><dl class="bkr_refwrap"><dt>3.2.6.</dt><dd id="ncipr42.lt20"><p class="no_top_margin">Reagent reservoirs</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr42.lt21"><p class="no_top_margin">Equipment
|
|
<dl id="ncipr42.l4" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.3.1.</dt><dd id="ncipr42.lt22"><p class="no_top_margin">Microcentrifuge</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.2.</dt><dd id="ncipr42.lt23"><p class="no_top_margin">Centrifuge capable of running at 2500xg, with a swinging basket set up to hold 5 cc vacutainer tubes</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.3.</dt><dd id="ncipr42.lt24"><p class="no_top_margin">Refrigerator, 2-8˚C</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.4.</dt><dd id="ncipr42.lt25"><p class="no_top_margin">Freezer, −20˚C</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.5.</dt><dd id="ncipr42.lt26"><p class="no_top_margin">Vortex</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.6.</dt><dd id="ncipr42.lt27"><p class="no_top_margin">Incubator, 37˚C</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.7.</dt><dd id="ncipr42.lt28"><p class="no_top_margin">ELISA plate reader (for single plex) capable of operating at 405 nm</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.8.</dt><dd id="ncipr42.lt29"><p class="no_top_margin">Q-View Imager Pro (for multi-plex) or similar imaging system</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr42.s4"><h2 id="_ncipr42_s4_">4. Reagent and Control Preparation</h2><dl id="ncipr42.l5" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr42.lt30"><p class="no_top_margin"><u>Positive Control 1 (Traditional substance known to activate complement)</u>
|
|
<dl id="ncipr42.l6" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.1.</dt><dd id="ncipr42.lt31"><p class="no_top_margin"><i>Cobra Venom Factor (CVF)</i> is supplied frozen solution. Thaw this stock, prepare single use aliquots and store them at a nominal temperature of −80˚C for as long as performance is acceptable. Avoid repeated freeze/thaw cycles. After thawing single use aliquot and using it in the assay, discard any leftover material. For this experiment, use 30 µL (1.1-50 U) of CVF solution. This control activates complement system through alternative pathway.</p></dd></dl><dl class="bkr_refwrap"><dt>4.1.2.</dt><dd id="ncipr42.lt32"><p class="no_top_margin"><i>Heat Aggregated Gamma Globulin (HAGG</i>) acts similarly to naturally occurring immune complexes and is very potent activator of complement through the classical pathway. This control is available from Quidel under name “Complement Activator”. Handling and storage are according to the manufacturer’s instructions. Avoid repeated freeze/thaw cycles when stored at −20°C.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>4.2.</dt><dd id="ncipr42.lt33"><p class="no_top_margin"><u>Positive Control 2 (nanoparticle relevant)</u>
|
|
<dl id="ncipr42.l7" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.2.1.</dt><dd id="ncipr42.lt34"><p class="no_top_margin"><u>Cremophor-EL</u></p><p>Cremophor-EL is an excipient commonly used in the pharmaceutical industry to dissolve hydrophobic drugs. Cremophor-EL is a nanosized micelle which is known to induce complement activation related pseudo allergy (CARPA) syndrome [<a class="bibr" href="#ncipr42.ref2" rid="ncipr42.ref2">2</a>], and therefore is used as a nanoparticle relevant control. The following procedure can be used to prepare Cremophor-EL with a composition that is similar to the clinical formulation of Paclitaxel (Taxol) (527 mg of purified Cremophor EL* (polyoxyethylated castor oil) and 49.7% (v/v) dehydrated alcohol, USP and 2 mg of citric acid per 1 mL). Store at room temperature. To prepare Cremophor-EL, mix commercial Cremophor 1:1 with ethanol containing 2 mg/mL of citric acid to mimic the concentration of Cremophor-EL, citric acid, and ethanol used in Taxol and the generic formulation of paclitaxel.</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.2.</dt><dd id="ncipr42.lt35"><p class="no_top_margin"><u>Cremophor-EL Formulated Paclitaxel (Taxol)</u></p><p>Taxol can be used as an alternative nanoparticle relevant positive control. It is supplied at a stock concentration 6 mg/mL of paclitaxel. When used in this assay, the final concentration of paclitaxel is 2 mg/mL. Store 2-8ºC.</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.3.</dt><dd id="ncipr42.lt36"><p class="no_top_margin"><u>PEGylated Liposomal Doxorubicin (Doxil)</u></p><p>Doxil can also be used as nanoparticle relevant positive control [<a class="bibr" href="#ncipr42.ref3" rid="ncipr42.ref3">3</a>]. Doxil is doxorubicin formulated in nanoliposomes. It is available through the pharmacy as 20 mg of Doxorubicin HCl in 10 mL vehicle. Store 2-8ºC.</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>4.3.</dt><dd id="ncipr42.lt37"><p class="no_top_margin">
|
|
<u>Inhibition/Enhancement Control (IEC)</u>
|
|
</p><p>Use positive control sample after the incubation. Prior to loading this sample onto ELISA plate, add nanoparticles at the same final concentrations as in the study samples. For example, one can mix 20 μL of the positive control sample and 10 μL of the test nanoparticle. The test result for this sample needs to be adjusted by the dilution factor 1.5 prior to comparison of the test value to the test value of the positive control sample. If the test results are different by no more than 25%, the test nanoparticle at the given concentration does not interfere with detection of the complement split product by ELISA.</p></dd></dl><dl class="bkr_refwrap"><dt>4.4.</dt><dd id="ncipr42.lt38"><p class="no_top_margin">
|
|
<u>Negative Control (PBS)</u>
|
|
</p><p>Sterile Ca<sup>2+</sup>/Mg<sup>2+</sup>-free PBS is used as a negative control. Store at room temperature for up to 6 months.</p></dd></dl><dl class="bkr_refwrap"><dt>4.5.</dt><dd id="ncipr42.lt39"><p class="no_top_margin">
|
|
<u>Vehicle Control (relevant to each given nanoparticle)</u>
|
|
</p><p>When nanoparticles are not formulated in saline or PBS, the vehicle sample should be tested to estimate the effect of excipients on the complement system. This control is specific to each given nanoparticle sample. It should be prepared to match the formulation buffer of the nanoparticle by both composition and concentration.</p></dd></dl><dl class="bkr_refwrap"><dt>4.5.</dt><dd id="ncipr42.lt40"><p class="no_top_margin">
|
|
<u>Stop Solution (HCl)</u>
|
|
</p><p>Stop solution is provided with each kit but can also be prepared separately. Dilute stock hydrochloric acid to a final concentration of 1.0 N. Filter and store and room temperature for up to 2 weeks.</p></dd></dl></dl></div><div id="ncipr42.s5"><h2 id="_ncipr42_s5_">5. Preparation of Study Samples</h2><p>This assay requires 400 µL of nanoparticles in PBS at a concentration 3 times higher than the highest final tested concentration. The concentration is selected based on the plasma concentration of the nanoparticle at the intended therapeutic dose. For the purpose of this protocol this concentration is called “theoretical plasma concentration”. Considerations for estimating theoretical plasma concentration were reviewed elsewhere [<a class="bibr" href="#ncipr42.ref4" rid="ncipr42.ref4">4</a>] and are summarized in <a href="/books/NBK604912/box/ncipr42.box8/?report=objectonly" target="object" rid-ob="figobncipr42box8">Box 1</a> below.</p><p>The assay will evaluate 4 concentrations: 10X (or when feasible 100X, 30X or 5X) of the theoretical plasma concentration, theoretical plasma concentration and two 1:5 serial dilutions of the theoretical plasma concentration. When the intended therapeutic concentration is unknown, the highest final concentration is 1 mg/mL or the highest reasonably achievable concentration.</p><p>For example, if the final theoretical plasma concentration to be tested is 0.2 mg/mL, then a stock of 6 mg/mL will be prepared and diluted 10-fold (0.6 mg/mL), followed by two 1:5 serial dilutions (0.12 and 0.024 mg/mL). When 0.1 mL of each of these samples is added to the test tube and mixed with 0.1 mL of plasma and 0.1 mL of veronal buffer, the final nanoparticle concentrations tested in the assay are: 2.0, 0.2, 0.04 and 0.008 mg/mL.</p><div id="ncipr42.box8" class="box boxed-text-box whole_rhythm hide-overflow"><h3><span class="label">Box 1</span><span class="title">Example Calculation to Determine Nanoparticle Concentration for In Vitro Tests</span></h3><p>In this example, we assume a mouse dose of 123 mg/kg. Therefore, the scaled equivalent human dose would be:
|
|
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr42.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr42.eq1" display="block"><mi>H</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi><mo>=</mo><mfrac><mrow><mi>m</mi><mi>o</mi><mi>u</mi><mi>s</mi><mi>e</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>123</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div>
|
|
Blood volume constitutes approximately 8% of the body weight. Therefore, an average human of 70 kg body weight has approximately 5.6 L of blood. Assuming all the nanoparticle injected goes into the systemic circulation, this provides a rough approximation of the potential maximum nanoparticle concentration in blood, which is used as the in vitro test concentration.</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr42.deq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
|
|
<math id="ncipr42.eq2" display="block"><mi>i</mi><mi>n</mi><mtext> </mtext><mi>v</mi><mi>i</mi><mi>t</mi><mi>r</mi><mi>o</mi><mtext> </mtext><mi>c</mi><mi>o</mi><mi>n</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>a</mi><mi>t</mi><mi>i</mi><mi>o</mi><msub><mi>n</mi><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>m</mi><mi>a</mi><mi>t</mi><mi>r</mi><mi>i</mi><mi>x</mi></mrow></msub><mo>=</mo><mfrac><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>b</mi><mi>l</mi><mi>o</mi><mi>o</mi><mi>d</mi><mtext> </mtext><mi>v</mi><mi>o</mi><mi>l</mi><mi>u</mi><mi>m</mi><mi>e</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>70</mn><mtext> </mtext><mi>k</mi><mi>g</mi><mtext> </mtext><mo>∗</mo><mtext> </mtext><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>5.6</mn><mtext> </mtext><mi>L</mi></mrow></mfrac><mo>=</mo><mn>0.125</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>m</mi><mi>L</mi></math>
|
|
</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></div></div><div id="ncipr42.s6"><h2 id="_ncipr42_s6_">6. Plasma Collection and Storage</h2><p>Blood is drawn into vacutainer tubes containing anticoagulant. Sodium citrate is ideal anti-coagulant for this assay, however depending on phlebotomy paraphernalia, plasma anti-coagulated with sodium citrate may result in high background in the ELISA assay. In this case using K<sub>2</sub>EDTA as the anticoagulant is acceptable. The first 5-10 mL of blood should be discarded and not used to prepare plasma. For optimal results, it is important to keep blood at 20-24ºC, to avoid exposure to high temperatures (summertime) and low temperatures (wintertime), and to avoid prolonged (> 1 hr) storage. Blood is transported to the lab in a contained Styrofoam box with warm packs (20-24°C). To prepare plasma, the blood is spun down in a centrifuge 10 minutes at 2500xg. Plasma is evaluated for the presence of hemolysis. Discolored plasma (an indication of hemolysis) is not used to prepare the pool. Individual plasma specimens that did not show any indication of hemolysis are pooled and mixed in a conical tube. Plasma must be used for complement testing within 1 hour after collection. Pooled plasma can be used and prepared by mixing plasma from at least 2 individual donors. The assay can also be performed in the plasma from individual donors. In this case analyze plasma from at least 3 donors.</p><p>It is possible to use pooled sodium citrate plasma from commercial suppliers, however, when placing the order, one needs to notify the supplier that the plasma is intended for complement testing so no delays between blood draw and plasma collection occurs. The supplier then freezes the plasma immediately after collection and ships it to the lab on dry ice. When using frozen plasma for the complement activation assay, it is important to avoid repeated freeze/thaw cycles. The frozen plasma should be thawed in a water bath containing ambient tap water, mixed gently and used immediately after thawing. It is also advised to avoid indefinite storage of frozen plasma at −20ºC. The sooner the frozen plasma is used, the better the results are. In general, the degree of complement activation estimated by comparing intensity of the C3 split product in the positive control with that of the negative control is greater in fresh plasma than in thawed plasma.</p></div><div id="ncipr42.s7"><h2 id="_ncipr42_s7_">7. Experimental Procedure for Sample Preparation</h2><dl id="ncipr42.l8" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>7.1.</dt><dd id="ncipr42.lt41"><p class="no_top_margin">In a microcentrifuge tube, combine equal volumes (100 µL of each) of veronal buffer, human plasma, and a test-sample (i.e., positive control, negative control, nanoparticles, or vehicle control if different than PBS). Prepare two replicates of each sample.</p></dd></dl><dl class="bkr_refwrap"><dt>7.2.</dt><dd id="ncipr42.lt42"><p class="no_top_margin">Vortex tubes to mix all reaction components, spin briefly in a microcentrifuge to bring any drops down, and incubate in an incubator at a nominal temperature of 37˚C for 30 minutes.</p></dd></dl><dl class="bkr_refwrap"><dt>7.3.</dt><dd id="ncipr42.lt43"><p class="no_top_margin">Prepare 100 µL aliquots and either use in EIA immediately or freeze at −20ºC for later analysis.</p></dd></dl></dl></div><div id="ncipr42.s8"><h2 id="_ncipr42_s8_">8. Experimental Procedure for Single-Plex EIA</h2><dl id="ncipr42.l9" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>8.1.</dt><dd id="ncipr42.lt44"><p class="no_top_margin">Follow the manufacturer’s instructions to reconstitute complement standard, wash buffers and controls.</p></dd></dl><dl class="bkr_refwrap"><dt>8.2.</dt><dd id="ncipr42.lt45"><p class="no_top_margin">Dilute plasma samples prepared in <a href="#ncipr42.lt43">step 7.3</a> in complement specimen diluent reagent (provided with each kit). Use the following dilution guide for each individual assay:
|
|
<ul id="ncipr42.l10" class="simple-list"><li id="ncipr42.lt46" class="half_rhythm"><div><b>iC3b –</b> 1:1500 for positive control sample; 1:30 for negative control and other test samples</div></li><li id="ncipr42.lt47" class="half_rhythm"><div><b>C4d –</b> 1:30 for all samples</div></li><li id="ncipr42.lt48" class="half_rhythm"><div><b>Bb –</b> 1:75 for all samples</div></li></ul>
|
|
<blockquote><p><i>Note: The dilution factors should be determined by each laboratory and adjusted if needed</i>.</p></blockquote></p></dd></dl><dl class="bkr_refwrap"><dt>8.3.</dt><dd id="ncipr42.lt49"><p class="no_top_margin">Follow manufacturer’s instructions for plate loading volumes, incubation time, and plate washing.</p></dd></dl><dl class="bkr_refwrap"><dt>8.4.</dt><dd id="ncipr42.lt50"><p class="no_top_margin">Read plate on a plate reader at 405 nm.</p></dd></dl></dl></div><div id="ncipr42.s9"><h2 id="_ncipr42_s9_">9. Experimental Procedure for Multi-plex ELISA</h2><dl id="ncipr42.l11" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>9.1.</dt><dd id="ncipr42.lt51"><p class="no_top_margin">Follow manufacturer’s instructions to reconstitute standards, wash buffer, and controls.</p></dd></dl><dl class="bkr_refwrap"><dt>9.2.</dt><dd id="ncipr42.lt52"><p class="no_top_margin">Dilute plasma samples prepared in <a href="#ncipr42.lt43">step 7.3</a> with sample diluent (provided with each kit). PC, NC, and all samples are diluted 1:100.</p></dd></dl><dl class="bkr_refwrap"><dt>9.3.</dt><dd id="ncipr42.lt53"><p class="no_top_margin">Follow manufacturer’s instructions for loading volumes, incubation time, and plate washing.</p></dd></dl><dl class="bkr_refwrap"><dt>9.4.</dt><dd id="ncipr42.lt54"><p class="no_top_margin">Capture a 300-second image of the plate on the Q-View Imager Pro or a 270-second image on the Q-View Imager LS.</p></dd></dl></dl></div><div id="ncipr42.s10"><h2 id="_ncipr42_s10_">10. Data Analysis</h2><p>Do not forget to use the appropriate dilution factor for control and study samples. Compare determined amount of complement components between positive control or study samples with that in the negative control. An increase in the complement component species 2.0-fold or higher above the background (negative control) constitutes a positive response. If a nanoparticle under study generated a positive response in any of the EIA assays, compare the degree of activation between this particle and the Doxil or other nanoparticle-relevant control. Doxil is used in the clinic and is known to induce complement activation related hypersensitivity reactions in sensitive patients [<a class="bibr" href="#ncipr42.ref5" rid="ncipr42.ref5">5</a>]. Using Doxil helps to interpret results of this <i>in vitro</i> study for a test nanoparticle. If the degree of activation observed for the test nanoparticle is equal to or greater than that observed for Doxil, this nanoparticle formulation will most likely cause similar or stronger hypersensitivity reactions in patients and may require modifications before entering <i>in vivo</i> preclinical and clinical phases. If the degree of activation is lower than that of Doxil, complement activation should be considered when designing the <i>in vivo</i> evaluation phase for the given particle, but it is less likely to cause concerns similar to Doxil.</p></div><div id="ncipr42.s11"><h2 id="_ncipr42_s11_">11. Acceptance Criteria</h2><dl id="ncipr42.l12" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>11.1.</dt><dd id="ncipr42.lt55"><p class="no_top_margin">Percent CV between replicates of standard curve, quality controls, and test samples should be within 25%.</p></dd></dl><dl class="bkr_refwrap"><dt>11.2.</dt><dd id="ncipr42.lt56"><p class="no_top_margin">Percent difference from theoretical for each of the standard curve samples should be within 25%, and correlation coefficient should be at or above 0.98.</p></dd></dl><dl class="bkr_refwrap"><dt>11.3.</dt><dd id="ncipr42.lt57"><p class="no_top_margin">Run is acceptable if conditions described in 11.1 and 11.2 are met.</p></dd></dl><dl class="bkr_refwrap"><dt>11.4.</dt><dd id="ncipr42.lt58"><p class="no_top_margin">The degree of complement activation in the positive control sample, estimated by comparing levels of individual complement split product in the positive control with that in the negative controls should be at or above 2.0-fold.</p><p>Note: Cobra venom factor activates complement through the alternative assay, so this control will not provide a positive response in the C4d assay. HAGG is a positive control for C4d assay. Doxil is positive in the C4d EIA.</p></dd></dl></dl></div><div id="ncipr42.rl.r1"><h2 id="_ncipr42_rl_r1_">12. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ncipr42.ref1">The Complement System. Complement reagents of the highest quality. EMD Biosciences, Calbiochem, Page 2. <a href="http://www.emdbiosciences.com/docs/docs/LIT/Complement_CB0617_EUSD.pdf" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>​.emdbiosciences<wbr style="display:inline-block"></wbr>​.com/docs/docs/LIT<wbr style="display:inline-block"></wbr>​/Complement_CB0617_EUSD.pdf</a></div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ncipr42.ref2">Weiszhár
|
|
Z, Czúcz
|
|
J, Révész
|
|
C, Rosivall
|
|
L, Szebeni
|
|
J, Rozsnyay
|
|
Z. Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, Tween-80 and Tween-20. Eur J Pharm Sci. 2012
|
|
Mar
|
|
12;45(4):492–8.
|
|
[<a href="https://pubmed.ncbi.nlm.nih.gov/21963457" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21963457</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ncipr42.ref3">Szebeni
|
|
J, Muggia
|
|
F, Gabizon
|
|
A, Barenholz
|
|
Y. Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention. Adv Drug Deliv Rev. 2011
|
|
Sep
|
|
16;63(12):1020–30
|
|
[<a href="https://pubmed.ncbi.nlm.nih.gov/21787819" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21787819</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ncipr42.ref4">Dobrovolskaia
|
|
MA, McNeil
|
|
SE. Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release. 2013;172(2):456–66.
|
|
[<a href="/pmc/articles/PMC5831149/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5831149</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23742883" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23742883</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ncipr42.ref5">Chanan-Khan
|
|
A, Szebeni
|
|
J, Savay
|
|
S, Liebes
|
|
L, Rafique
|
|
NM, Alving
|
|
CR, Muggia
|
|
FM. Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions. Ann Oncol. 2003, 14, 1430–1437.
|
|
[<a href="https://pubmed.ncbi.nlm.nih.gov/12954584" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12954584</span></a>]</div></dd></dl></dl></div><div id="ncipr42.s12"><h2 id="_ncipr42_s12_">13. Abbreviations</h2><dl><dt id="ncipr42.abb_DL1_DI1">CVF</dt><dd><p>cobra venom factor</p></dd><dt id="ncipr42.abb_DL1_DI2">PBS</dt><dd><p>phosphate buffered saline</p></dd><dt id="ncipr42.abb_DL1_DI3">EIA</dt><dd><p>enzyme immunoassay</p></dd><dt id="ncipr42.abb_DL1_DI4">HAGG</dt><dd><p>heat aggregated gamma globulin</p></dd><dt id="ncipr42.abb_DL1_DI5">HRP</dt><dd><p>horseradish peroxidase</p></dd><dt id="ncipr42.abb_DL1_DI6">IEC</dt><dd><p>inhibition/enhancement control</p></dd><dt id="ncipr42.abb_DL1_DI7">IgG (H + L)</dt><dd><p>immunoglobulin G (high and low chains)</p></dd><dt id="ncipr42.abb_DL1_DI8">NC</dt><dd><p>negative control</p></dd><dt id="ncipr42.abb_DL1_DI9">PC</dt><dd><p>positive control</p></dd></dl></div><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ncipr42.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="ncipr42.suggestedcitation">Neun B, Cedrone E, Dobrovolskaia MA, NCL Method ITA-5.2: Analysis of Complement Activation by single-plex EIA or multiplex ELISA. <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/EQCT-3C51" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/EQCT-3C51</a></span>
|
|
</div></p></div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK604912_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Barry W. Neun</span>, <span itemprop="author">Edward Cedrone</span>, 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>Contact</h4><div class="affiliation"><sup>1</sup> <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 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><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">May 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>Neun BW, Cedrone E, Dobrovolskaia MA. Analysis of Complement Activation by Single-Plex EIA or Multiplex ELISA: Version 3. 2020 May. In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method ITA-5.2.<span class="bk_cite_avail"></span> doi: 10.17917/EQCT-3C51</p></div><div class="small-screen-prev"><a href="/books/n/nciprotocols/ncipr44/?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/ncipr39/?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="figobncipr42fig1"><div id="ncipr42.fig1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Complement%20activation%20pathways.&p=BOOKS&id=604912_ncipr42f1.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/NBK604912/bin/ncipr42f1.jpg" alt="Figure 1. Complement activation pathways." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Complement activation pathways</span></h3><div class="caption"><p>(This illustration is reproduced from <a class="bibr" href="#ncipr42.ref1" rid="ncipr42.ref1">reference 1</a> with permission from EMD Biosciences, Inc.)</p></div></div></article><article data-type="boxed-text" id="figobncipr42box8"><div id="ncipr42.box8" class="box boxed-text-box whole_rhythm hide-overflow"><h3><span class="label">Box 1</span><span class="title">Example Calculation to Determine Nanoparticle Concentration for In Vitro Tests</span></h3><p>In this example, we assume a mouse dose of 123 mg/kg. Therefore, the scaled equivalent human dose would be:
|
|
<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr42.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col"><math id="ncipr42.eq1" display="block"><mi>H</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi><mo>=</mo><mfrac><mrow><mi>m</mi><mi>o</mi><mi>u</mi><mi>s</mi><mi>e</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>123</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>12.3</mn></mrow></mfrac><mo>=</mo><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div>
|
|
Blood volume constitutes approximately 8% of the body weight. Therefore, an average human of 70 kg body weight has approximately 5.6 L of blood. Assuming all the nanoparticle injected goes into the systemic circulation, this provides a rough approximation of the potential maximum nanoparticle concentration in blood, which is used as the in vitro test concentration.</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr42.deq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
|
|
<math id="ncipr42.eq2" display="block"><mi>i</mi><mi>n</mi><mtext> </mtext><mi>v</mi><mi>i</mi><mi>t</mi><mi>r</mi><mi>o</mi><mtext> </mtext><mi>c</mi><mi>o</mi><mi>n</mi><mi>c</mi><mi>e</mi><mi>n</mi><mi>t</mi><mi>r</mi><mi>a</mi><mi>t</mi><mi>i</mi><mi>o</mi><msub><mi>n</mi><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>m</mi><mi>a</mi><mi>t</mi><mi>r</mi><mi>i</mi><mi>x</mi></mrow></msub><mo>=</mo><mfrac><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>d</mi><mi>o</mi><mi>s</mi><mi>e</mi></mrow><mrow><mi>h</mi><mi>u</mi><mi>m</mi><mi>a</mi><mi>n</mi><mtext> </mtext><mi>b</mi><mi>l</mi><mi>o</mi><mi>o</mi><mi>d</mi><mtext> </mtext><mi>v</mi><mi>o</mi><mi>l</mi><mi>u</mi><mi>m</mi><mi>e</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><mn>70</mn><mtext> </mtext><mi>k</mi><mi>g</mi><mtext> </mtext><mo>∗</mo><mtext> </mtext><mn>10</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>k</mi><mi>g</mi></mrow><mrow><mn>5.6</mn><mtext> </mtext><mi>L</mi></mrow></mfrac><mo>=</mo><mn>0.125</mn><mtext> </mtext><mi>m</mi><mi>g</mi><mo>/</mo><mi>m</mi><mi>L</mi></math>
|
|
</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></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><script type="text/javascript" src="/core/mathjax/2.7.9/MathJax.js?config=TeX-AMS-MML_SVG"> </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 portal107 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>
|