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<p><strong>Enzyme-Linked ImmunoSorbent Assay</strong>, or <strong>ELISA</strong>, is a <a title="Biochemistry" href="http://en.wikipedia.org/wiki/Biochemistry" title="Biochemistry">biochemical</a> technique used mainly in <a title="Immunology" href="http://en.wikipedia.org/wiki/Immunology" title="Immunology">immunology</a> to detect the presence of an <a title="Antibody" href="http://en.wikipedia.org/wiki/Antibody" title="Antibody">antibody</a> or an <a title="Antigen" href="http://en.wikipedia.org/wiki/Antigen" title="Antigen">antigen</a> in a sample. The ELISA has been used as a <a title="Diagnosis" href="http://en.wikipedia.org/wiki/Diagnosis" title="Diagnosis">diagnostic</a> tool in medicine and <a title="Plant pathology" class="mw-redirect" href="http://en.wikipedia.org/wiki/Plant_pathology" class="mw-redirect" title="Plant pathology">plant pathology</a>, as well as a <a title="Quality control" href="http://en.wikipedia.org/wiki/Quality_control" title="Quality control">quality control</a> check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light is shone upon the sample, any antigen/antibody complexes will <a title="Fluorescence" href="http://en.wikipedia.org/wiki/Fluorescence" title="Fluorescence">fluoresce</a> so that the amount of antigen in the sample can be measured.</p><p>Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a <a title="Polystyrene" href="http://en.wikipedia.org/wiki/Polystyrene" title="Polystyrene">polystyrene</a> microtiter plate) either non-specifically (via <a title="Adsorption" href="http://en.wikipedia.org/wiki/Adsorption" title="Adsorption">adsorption</a> to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an <a title="Enzyme" href="http://en.wikipedia.org/wiki/Enzyme" title="Enzyme">enzyme</a>, or can itself be detected by a <a title="Secondary antibody" href="http://en.wikipedia.org/wiki/Secondary_antibody" title="Secondary antibody">secondary antibody</a> which is linked to an enzyme through <a title="Bioconjugation" href="http://en.wikipedia.org/wiki/Bioconjugation" title="Bioconjugation">bioconjugation</a>. Between each step the plate is typically washed with a mild <a title="Detergent" href="http://en.wikipedia.org/wiki/Detergent" title="Detergent">detergent</a> solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic <a title="Substrate" href="http://en.wikipedia.org/wiki/Substrate" title="Substrate">substrate</a> to produce a visible <a title="Signal (information theory)" class="mw-redirect" href="http://en.wikipedia.org/wiki/Signal_%28information_theory%29" class="mw-redirect" title="Signal (information theory)">signal</a>, which indicates the quantity of antigen in the sample. Older ELISAs utilize <a title="Chromogenic" href="http://en.wikipedia.org/wiki/Chromogenic" title="Chromogenic">chromogenic</a> substrates, though newer assays employ <a title="Fluorogenic" href="http://en.wikipedia.org/wiki/Fluorogenic" title="Fluorogenic">fluorogenic</a> substrates with much higher sensitivity.</p>
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<div class="thumbinner" style="width: 302px;" class="thumbinner"><a title="A 96-well microtiter plate being used for ELISA." class="image" href="http://en.wikipedia.org/wiki/Image:Microtiter_plate.JPG" class="image" title="A 96-well microtiter plate being used for ELISA."><img width="300" height="225" border="0" classalt="thumbimageA 96-well microtiter plate being used for ELISA." src="http://upload.wikimedia.org/wikipedia/commons/thumb/b/ba/Microtiter_plate.JPG/300px-Microtiter_plate.JPG" altclass="A 96-well microtiter plate being used for ELISA.thumbimage" /></a>
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<div class="magnify"><a title="Enlarge" class="internal" href="http://en.wikipedia.org/wiki/Image:Microtiter_plate.JPG" class="internal" title="Enlarge"><img width="15" height="11" alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" alt="" /></a></div>A 96-well <a title="Microtiter plate" href="http://en.wikipedia.org/wiki/Microtiter_plate" title="Microtiter plate">microtiter plate</a> being used for ELISA.</div>
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<p><strong>Enzyme ImmunoAssay (EIA)</strong> is a synonym for the ELISA.</p>
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<p><a idname="Applications" nameid="Applications"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">Applications</span></h2>
<p>Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining <a title="Blood plasma" href="http://en.wikipedia.org/wiki/Blood_plasma" title="Blood plasma">serum</a> antibody concentrations (such as with the <a title="HIV test" href="http://en.wikipedia.org/wiki/HIV_test" title="HIV test">HIV test</a><sup class="reference" id="_ref-0" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-0" title="">[1]</a></sup> or <a title="West Nile Virus" class="mw-redirect" href="http://en.wikipedia.org/wiki/West_Nile_Virus" class="mw-redirect" title="West Nile Virus">West Nile Virus</a>) and also for detecting the presence of antigen. It has also found applications in the <a title="Food" href="http://en.wikipedia.org/wiki/Food" title="Food">food</a> industry in detecting potential <a title="Food allergy" href="http://en.wikipedia.org/wiki/Food_allergy" title="Food allergy">food allergens</a> such as <a title="Milk" href="http://en.wikipedia.org/wiki/Milk" title="Milk">milk</a>, <a title="Peanut" href="http://en.wikipedia.org/wiki/Peanut" title="Peanut">peanuts</a>, <a title="Walnut" href="http://en.wikipedia.org/wiki/Walnut" title="Walnut">walnuts</a>, <a title="Almond" href="http://en.wikipedia.org/wiki/Almond" title="Almond">almonds</a>, and <a title="Egg (food)" href="http://en.wikipedia.org/wiki/Egg_%28food%29" title="Egg (food)">eggs</a>.<sup class="reference" id="_ref-1" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-1" title="">[2]</a></sup> The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.In an ELISA test, a person's serum is diluted 400-fold and applied to a plate to which HIV antigens have been attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to human antibodies — is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme. Thus the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and negative result.</p><p><a idname="History" nameid="History"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">History</span></h2>
<p>Prior to the development of the EIA/ELISA, the only option for conducting an <a title="Immunoassay" href="http://en.wikipedia.org/wiki/Immunoassay" title="Immunoassay">immunoassay</a> was <a title="Radioimmunoassay" href="http://en.wikipedia.org/wiki/Radioimmunoassay" title="Radioimmunoassay">radioimmunoassay</a>, a technique using <a title="Radioactive" class="mw-redirect" href="http://en.wikipedia.org/wiki/Radioactive" class="mw-redirect" title="Radioactive">radioactively</a>-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by <a title="Rosalyn Sussman Yalow" href="http://en.wikipedia.org/wiki/Rosalyn_Sussman_Yalow" title="Rosalyn Sussman Yalow">Rosalyn Sussman Yalow</a> and <a title="Solomon Berson" href="http://en.wikipedia.org/wiki/Solomon_Berson" title="Solomon Berson">Solomon Berson</a> published in 1960<sup class="reference" id="_ref-2" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-2" title="">[3]</a></sup>.</p><p>Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as <a title="Peroxidase" href="http://en.wikipedia.org/wiki/Peroxidase" title="Peroxidase">peroxidase</a>) react with appropriate substrates (such as <a title="ABTS" href="http://en.wikipedia.org/wiki/ABTS" title="ABTS">ABTS</a> or <a title="3,3’,5,5’-Tetramethylbenzidine" href="http://en.wikipedia.org/wiki/3%2C3%E2%80%99%2C5%2C5%E2%80%99-Tetramethylbenzidine" title="3,3’,5,5’-Tetramethylbenzidine">3,3’,5,5’-Tetramethylbenzidine</a>), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce<sup class="reference" id="_ref-3" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-3" title="">[4]</a></sup>. Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the <em>immunosorbent</em> has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966<sup class="reference" id="_ref-4" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-4" title="">[5]</a></sup></p><p>In 1971, Peter Perlmann and <a title="Eva Engvall" href="http://en.wikipedia.org/wiki/Eva_Engvall" title="Eva Engvall">Eva Engvall</a> at Stockholm University in Sweden, as well as Anton Schuurs and Bauke van Weemen in The Netherlands, independently published papers which synthesized this knowledge into methods to perform EIA/ELISA<sup class="reference" id="_ref-5" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-5" title="">[6]</a></sup><sup class="reference" id="_ref-6" class="reference"><a title="" href="http://en.wikipedia.org/wiki/ELISA#_note-6" title="">[7]</a></sup></p><p><a idname="Types" nameid="Types"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">Types</span></h2>
<p><a name=".22indirect.22_ELISA"></a></p>
<p>The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:</p>
<ol>
<li>Apply a sample of known antigen of known concentration to a surface, often the well of a <a title="Microtiter plate" href="http://en.wikipedia.org/wiki/Microtiter_plate" title="Microtiter plate">microtiter plate</a>. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a <a title="Standard curve" href="http://en.wikipedia.org/wiki/Standard_curve" title="Standard curve">standard curve</a> used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.</li> <li>The plate wells or other surface are then coated with <a title="Blood plasma" href="http://en.wikipedia.org/wiki/Blood_plasma" title="Blood plasma">serum</a> samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.</li> <li>A concentrated solution of non-interacting protein, such as <a title="Bovine Serum Albumin" class="mw-redirect" href="http://en.wikipedia.org/wiki/Bovine_Serum_Albumin" class="mw-redirect" title="Bovine Serum Albumin">Bovine Serum Albumin</a> (BSA) or <a title="Casein" href="http://en.wikipedia.org/wiki/Casein" title="Casein">casein</a>, is added to all plate wells. This step is known as <a title="Blocking" href="http://en.wikipedia.org/wiki/Blocking" title="Blocking">blocking</a>, because the serum proteins block non-specific adsorption of other proteins to the plate.</li>
<li>The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.</li>
<li>The plate is washed to remove any unbound detection antibody. After this wash, only the antibody-antigen complexes remain attached to the well.</li>
<p>The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich ELISA provides a solution to this problem.</p>
<p>ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.</p>
<p><a idname="Sandwich_ELISA" nameid="Sandwich_ELISA"></a></p>
<h4><span class="editsection"></span><span class="mw-headline">Sandwich ELISA</span></h4>
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<div class="thumbinner" style="width: 302px;"><a href="http://en.wikipedia.org/wiki/Image:ELISA-sandwich.svg" class="thumbinnerimage"><a title="A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form." class="image" href="http://en.wikipedia.org/wiki/Image:ELISA-sandwich.svg"><img width="300" height="150" border="0" class="thumbimage" src="http://upload.wikimedia.org/wikipedia/commons/thumb/4/4b/ELISA-sandwich.svg/300px-ELISA-sandwich.svg.png" alt="A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form." src="http://upload.wikimedia.org/wikipedia/commons/thumb/4/4b/ELISA-sandwich.svg/300px-ELISA-sandwich.svg.png" class="thumbimage" /></a>
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<div class="magnify"><a title="Enlarge" class="internal" href="http://en.wikipedia.org/wiki/Image:ELISA-sandwich.svg" class="internal" title="Enlarge"><img width="15" height="11" alt="" src="http://en.wikipedia.org/skins-1.5/common/images/magnify-clip.png" alt="" /></a></div>
<strong>A sandwich <em>ELISA</em></strong>. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.</div>
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<p>The image to the right includes the use of a secondary antibody conjugated to an enzyme, though technically this is not necessary if the capture antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. The major advantage of a sandwich ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.</p>
<p><a idname="Competitive_ELISA" nameid="Competitive_ELISA"></a></p><h4><span class="editsection">[<a title="Edit section: Competitive ELISA" href="http://en.wikipedia.org/w/index.php?title=ELISA&action=edit&section=6" title="Edit section: Competitive ELISA">edit</a>]</span> <span class="mw-headline">Competitive ELISA</span></h4>
<p>A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:</p>
<ol>
<li>These bound antibody/antigen complexes are then added to an antigen coated well.</li>
<li>The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")</li>
<li>The <a title="Secondary antibody" href="http://en.wikipedia.org/wiki/Secondary_antibody" title="Secondary antibody">secondary antibody</a>, specific to the primary antibody is added. This second antibody is coupled to the enzyme.</li>
<li>A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.</li>
</ol>
<p>For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.</p>
<p>(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal.)</p>
<p><a idname="See_also" nameid="See_also"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">See also</span></h2>
<ul>
<li><a title="Assay" href="http://en.wikipedia.org/wiki/Assay" title="Assay">Assay</a></li> <li><a title="Eva Engvall" href="http://en.wikipedia.org/wiki/Eva_Engvall" title="Eva Engvall">Eva Engvall</a></li> <li><a title="ELISPOT" href="http://en.wikipedia.org/wiki/ELISPOT" title="ELISPOT">ELISPOT</a></li> <li><a title="Immunoassay" href="http://en.wikipedia.org/wiki/Immunoassay" title="Immunoassay">Immunoassay</a></li> <li><a title="Radioimmunoassay" href="http://en.wikipedia.org/wiki/Radioimmunoassay" title="Radioimmunoassay">Radioimmunoassay</a></li> <li><a title="Secretion assay" href="http://en.wikipedia.org/wiki/Secretion_assay" title="Secretion assay">Secretion assay</a></li> <li><a title="Lateral flow test" href="http://en.wikipedia.org/wiki/Lateral_flow_test" title="Lateral flow test">Lateral flow test</a></li>
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<p><a idname="External_links" nameid="External_links"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">External links</span></h2>
<ul>
<li><a rel="nofollow" titlehref="http://www.elispot-analyzers.de/english/elisa-animation.html" class="external text" hreftitle="http://www.elispot-analyzers.de/english/elisa-animation.html" rel="nofollow">An animated illustration of an ELISA assay</a></li> <li><a rel="nofollow" titlehref="http://www.sumanasinc.com/webcontent/animations/content/ELISA.html" class="external text" hreftitle="http://www.sumanasinc.com/webcontent/animations/content/ELISA.html" rel="nofollow">An animated tutorial comparing the direct and indirect ELISA methods</a></li> <li><a rel="nofollow" titlehref="http://www.prosci-inc.com/ELISA-Protocol.html" class="external text" hreftitle="http://www.prosci-inc.com/ELISA-Protocol.html" rel="nofollow">ELISA Protocol</a></li> <li><a rel="nofollow" titlehref="http://www.biology.arizona.edu/immunology/activities/elisa/main.html" class="external text" hreftitle="http://www.biology.arizona.edu/immunology/activities/elisa/main.html" rel="nofollow">"Introduction to ELISA Activity - beginner walkthrough of ELISA used for detecting HIV, including animations</a> at <a title="University of Arizona" href="http://en.wikipedia.org/wiki/University_of_Arizona" title="University of Arizona">University of Arizona</a></li> <li><a title="Medical Subject Headings" href="http://en.wikipedia.org/wiki/Medical_Subject_Headings" title="Medical Subject Headings">MeSH</a> <em><a rel="nofollow" titlehref="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&term=ELISA" class="external text" hreftitle="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&term=ELISA" rel="nofollow">ELISA</a></em></li>
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<p><a idname="References" nameid="References"></a></p>
<h2><span class="editsection"></span><span class="mw-headline">References</span></h2>
<div class="references-small" style="-moz-column-count: 2;" class="references-small">
<ol class="references">
<li id="_note-0"><strong><a title="" href="http://en.wikipedia.org/wiki/ELISA#_ref-0" title="">^</a></strong> MedLinePlus. "HIV ELISA/western blot." U.S. National Library of Medicine. Last accessed April 16, 2007. <a rel="nofollow" titlehref="http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm" class="external free" hreftitle="http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm" rel="nofollow">http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm</a></li> <li id="_note-1"><strong><a title="" href="http://en.wikipedia.org/wiki/ELISA#_ref-1">title="">^</a></strong> U. S. Food and Drug Administration. "Food Allergen Partnership." Last accessed April 16, 2007. <a rel="nofollow" titlehref="http://www.cfsan.fda.gov/~dms%7Edms/alrgpart.html" class="external free" hreftitle="http://www.cfsan.fda.gov/%7Edms~dms/alrgpart.html" rel="nofollow">http://www.cfsan.fda.gov/~dms/alrgpart.html</a></li> <li id="_note-2"><strong><a title="" hrefhref="http://en.wikipedia.org/wiki/ELISA#_ref-2" title="">^</a></strong> <cite style="font-style: normal;">YALOW R, BERSON S (1960). "Immunoassay of endogenous plasma insulin in man". <em>J. Clin. Invest.</em> 39: 1157-75. <a titlehref="http://www.ncbi.nlm.nih.gov/pubmed/13846364" class="external" hreftitle="http://www.ncbi.nlm.nih.gov/pubmed/13846364">PMID 13846364</a>.</cite><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Immunoassay+of+endogenous+plasma+insulin+in+man&rft.jtitle=J.+Clin.+Invest.&rft.date=1960&rft.volume=39&rft.au=YALOW+R%2C+BERSON+S&rft.pages=1157-75&rft_id=info:pmid/13846364" class="Z3988"> </span></li> <li id="_note-3"><strong><a title="" href="http://en.wikipedia.org/wiki/ELISA#_ref-3">title="">^</a></strong> <cite style="font-style: normal;">Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". <em>Clin. Chem.</em> 51 (12): 2415-8. <a titlehref="http://www.ncbi.nlm.nih.gov/pubmed/16179424" class="external" hreftitle="http://www.ncbi.nlm.nih.gov/pubmed/16179424">PMID 16179424</a>.</cite><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Enzyme+immunoassay+%28EIA%29%2Fenzyme-linked+immunosorbent+assay+%28ELISA%29.&rft.jtitle=Clin.+Chem.&rft.date=2005&rft.volume=51&rft.issue=12&rft.au=Lequin+R&rft.pages=2415-8&rft_id=info:pmid/16179424" class="Z3988"> </span></li> <li id="_note-4"><strong><a title="" href="http://en.wikipedia.org/wiki/ELISA#_ref-4" title="">^</a></strong> Wide L, Porath J. Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies. Biochem Biophys Acta 1966;30:257-260.</li> <li id="_note-5"><strong><a title="" hrefhref="http://en.wikipedia.org/wiki/ELISA#_ref-5" title="">^</a></strong> <cite style="font-style: normal;">Engvall E, Perlman P (1971). "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". <em>Immunochemistry</em> 8 (9): 871-4. <a titlehref="http://www.ncbi.nlm.nih.gov/pubmed/5135623" class="external" hreftitle="http://www.ncbi.nlm.nih.gov/pubmed/5135623">PMID 5135623</a>.</cite><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Enzyme-linked+immunosorbent+assay+%28ELISA%29.+Quantitative+assay+of+immunoglobulin+G&rft.jtitle=Immunochemistry&rft.date=1971&rft.volume=8&rft.issue=9&rft.au=Engvall+E%2C+Perlman+P&rft.pages=871-4&rft_id=info:pmid/5135623" class="Z3988"> </span></li> <li id="_note-6"><strong><a title="" href="http://en.wikipedia.org/wiki/ELISA#_ref-6" title="">^</a></strong> <cite style="font-style: normal;">Van Weemen BK, Schuurs AH (1971). "Immunoassay using antigen-enzyme conjugates.". <em>FEBS Letters</em> 15 (3): 232-6. <a titlehref="http://www.ncbi.nlm.nih.gov/pubmed/11945853" class="external" hreftitle="http://www.ncbi.nlm.nih.gov/pubmed/11945853">PMID 11945853</a>.</cite><span class="Z3988" title="ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Immunoassay+using+antigen-enzyme+conjugates.&rft.jtitle=FEBS+Letters&rft.date=1971&rft.volume=15&rft.issue=3&rft.au=Van+Weemen+BK%2C+Schuurs+AH&rft.pages=232-6&rft_id=info:pmid/11945853" class="Z3988"> </span></li>
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