{"id":10415,"date":"2023-08-03T04:35:23","date_gmt":"2023-08-03T08:35:23","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=10415"},"modified":"2024-02-01T04:02:13","modified_gmt":"2024-02-01T09:02:13","slug":"sandwich-elisa","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/sandwich-elisa\/","title":{"rendered":"Assay Setup: Sandwich ELISA for Allergy"},"content":{"rendered":"\n\n\n\t<div class=\"dkpdf-button-container\" style=\" text-align:right \">\n\n\t\t<a class=\"dkpdf-button\" href=\"\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10415?pdf=10415\" target=\"_blank\"><span class=\"dkpdf-button-icon\"><i class=\"fa fa-file-pdf-o\"><\/i><\/span> Download PDF<\/a>\n\n\t<\/div>\n\n\n\n\n\n<p><style>.entry p, .entry ol{font-size: 1rem;}.entry h3{color:#009fe3;margin-top:0}.entry h4{color:#009453;margin-top:1.5rem}.entry h5{color: #003a5c; font-size: 1.05rem;margin-top:1.5rem;}.entry figure{margin:32px auto;border:1px solid #ccc}.entry figure img{width:100%;display:block;margin:0 auto}.entry figcaption{font-size:.875rem;line-height:1.35rem;padding:10px;color:#222}.entry .blog-tbl{margin:1rem auto;caption-side:bottom}.entry .blog-tbl td,.entry .blog-tbl th{padding:10px 9px;border:1px solid #00172b;text-align:left}.entry .blog-tbl td{border-color:#003a5c}.entry .blog-tbl th{background-color:#00172b;color:#fff;border-left-color:#fff;border-right-color:#fff}.entry .blog-tbl th p{color:#fff}.entry .blog-tbl th:first-of-type{border-left-color:#00172b}.entry .blog-tbl th:last-of-type{border-right-color:#00172b}.entry .blog-tbl p{text-align:left;margin:0}.entry .box-note{border:2px solid #009453;padding:12px;margin:1rem 0}.entry .box-note p{margin:0;padding:0}.entry .styled-list{list-style-type:none}.entry .styled-list li{margin-top:1rem;line-height:1rem;font-size:1rem;}.entry .styled-list li::before{font-family:\"Font Awesome 5 Pro\";display:inline-block;content:\"\\f3c5\";-webkit-transform:rotate(-90deg);transform:rotate(-90deg);margin-left:-20px;margin-right:11px;font-size:.75rem;color:#ed7004;font-weight:600}.entry .styled-list ol li::before{display:none}.entry .styled-list li>ul li::before{font-weight:200}.entry .overview{width:-webkit-fit-content;width:-moz-fit-content;width:fit-content;padding:16px;margin:1rem auto;border:1px solid #eee}.entry .overview hr{margin-top:14px}.entry .overview-text{text-align:center;font-size:1rem;margin:0}.entry .btn-sq{color:#fff;font-size:.9rem;font-weight:600;border:2px solid rgb(237, 112, 4);padding:7px 13px;background:rgb(237, 112, 4);cursor:pointer;text-align:center;line-height:1.5rem;margin:0 auto;}.entry .btn-sq:hover{text-decoration:none;color:rgb(237, 112, 4);background:#fff;}.entry .btn-container{display:flex;width:100%;margin:1.3rem 0;}.entry .btn-container .fa-solid, .entry .btn-container .fa-duotone{margin-right:10px;}@media(max-width: 768px){.entry .btn-sq{font-size:1.05rem;}}<\/style> <style>.entry .styled-list li::before{display:none} .entry .styled-list li{list-style-type:decimal;line-height:24px}.entry .styled-list li::marker{font-size:75%}.entry figure{max-width: 500px}.entry .btn-container{flex-wrap: wrap}.entry .btn-sq:nth-child(5){margin-top:2rem}@media (max-width:624px){.entry .bottom-btns .btn-sq:not(.entry .bottom-btns .btn-sq:first-child){margin-top: 2rem}}.entry .box-note{background:#e6ffdf}.entry .blog-tbl td{background:#fff;}<\/style><\/p>\n<p><script src=\"https:\/\/kit.fontawesome.com\/904923013f.js\" crossorigin=\"anonymous\"><\/script><\/p>\n<h2><span style=\"font-weight: 400;\">ELISA is a widely used technique for detecting and quantifying one or more specific proteins within a complex mixture. ELISAs are quick and easy to perform and can be adapted to detect almost any analyte, offering a highly sensitive technique that can be used to process large sample numbers. ELISAs can be performed in a number of formats, but Sandwich ELISA is the most commonly used, with almost unlimited utility for basic research as well as for clinical applications like disease diagnosis and health monitoring. This format uses matched antibody pairs for capture and detection. Here we describe how to set up a sandwich ELISA for optimum sensitivity and specificity.\u00a0<\/span><\/h2>\n<p><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10441 size-full\" style=\"width: 100%;\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/ELISA-4-1.jpg\" alt=\"\" width=\"600\" height=\"229\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/ELISA-4-1.jpg 600w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/ELISA-4-1-300x115.jpg 300w\" sizes=\"(max-width: 600px) 100vw, 600px\" \/><\/p>\n<p><span style=\"font-weight: 400;\">Sandwich ELISA is the most commonly-used ELISA format, which uses matched antibody pairs for capture and detection to enable specificity and sensitivity for the analyte of interest from complex mixtures such as biological samples. The <\/span><span style=\"font-weight: 400;\">Sandwich format enables superior specificity compared to either a direct or indirect ELISA because there are two distinct analyte-binding antibodies. Each of the antibodies are able to recognize a different antigenic epitope, preventing competition.\u00a0<\/span><\/p>\n<hr \/>\n<h3><span style=\"font-weight: 400;\">Set up<\/span><\/h3>\n<p><span style=\"font-weight: 400;\">The sandwich format may be performed directly or indirectly. It uses an analyte-specific capture antibody which is bound directly to microplate wells and captures the analyte from the sample. The sample is added to the well, and the plate-bound antibody captures the analyte from this sample. A second analyte-specific \u201cprimary\u201d antibody, which is also specific to the analyte, is then added. This antibody may or may not be labeled with a reporter molecule, depending on whether it is a direct or indirect sandwich. If the \u201cPrimary antibody\u201d is unlabeled, then a third \u201cDetection\u201d or \u201cSecondary\u201d antibody is added. Conjugated to a reporter enzyme or a fluorescent probe, it acts to amplify the achievable signal.<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10445 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-3-100.jpg\" alt=\"\" width=\"641\" height=\"640\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-3-100.jpg 641w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-3-100-300x300.jpg 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-3-100-150x150.jpg 150w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-3-100-45x45.jpg 45w\" sizes=\"(max-width: 641px) 100vw, 641px\" \/>\n<figcaption><strong>Figure 1:<\/strong> Sandwich ELISA benefits from superior specificity compared to either a direct or indirect ELISA because two distinct analyte-binding antibodies (each recognizing a different antigenic epitope) are used. It may also provide greater sensitivity, and even low-abundance antigens can be detected within complex samples such as serum.<\/figcaption>\n<\/figure>\n<hr \/>\n<h3><span style=\"font-weight: 400;\">The indirect sandwich ELISA:\u00a0<\/span><\/h3>\n<h4><span style=\"font-weight: 400;\">Polyclonal or Monoclonal?<\/span><\/h4>\n<p><span style=\"font-weight: 400;\">The clonality of the antibodies chosen can impact the performance of the assay. Monoclonals can offer consistent lot-to-lot performance and guaranteed long-term supply, whereas polyclonals can increase the likelihood of target detection and improve sensitivity.\u00a0<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10446 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/poly-mono-amplification-1.jpg\" alt=\"\" width=\"487\" height=\"178\" \/>\n<p>\u00a0<\/p>\n<figcaption><strong>Figure 2:<\/strong> Monoclonal antibodies are comprised of one immunoglobulin clone which detects a single antigenic epitope, making the antibody very specific but limiting it to one site. Polyclonal antibodies are made up of a heterogenous population of immunoglobulins which detect a variety of epitopes and can bind multiple sites across the target.<\/figcaption>\n<\/figure>\n<p><span style=\"font-weight: 400;\">Learn more <\/span><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/elisa-guide-part-2\/\"><span style=\"font-weight: 400;\">here<\/span><\/a><\/p>\n<p><span style=\"font-weight: 400;\">Some assays may incorporate a monoclonal antibody for antigen capture and a polyclonal antibody for detection, or they may use a pair of monoclonal antibodies. If the sandwich ELISA relies on using matched monoclonal antibody pairs, it is important that they have been rigorously validated for the ELISA application.\u00a0<\/span><\/p>\n<h4><span style=\"font-weight: 400;\">Validate antibodies<\/span><\/h4>\n<p><span style=\"font-weight: 400;\">If using monoclonal antibodies it is critical to prevent the two antibodies from competing with one another to bind the target epitope. Performing control experiments ensures that the analyte-specific antibodies can detect the antigen under the conditions of the ELISA, where proteins may be in their native forms rather than those used to confirm reactivity by Western blot. Competition assays may be used to confirm that two different epitopes are targeted by the antibody pairs.\u00a0<\/span><\/p>\n<hr \/>\n<div class=\"box-note\">\n<h3><span style=\"font-weight: 400;\">If using polyclonal antibodies, choose min x to reduce off-target signal<\/span><\/h3>\n<p><span style=\"font-weight: 400;\">Experiments may need a combination of monoclonal and polyclonal antibodies, which will require the experimental design to consider the impact of host and sample species, and any blocking reagents that can potentially cross-react, generating unwanted background signal by introducing non-specific binding. Choosing an antibody from Jackson ImmunoResearch that is minimally cross-reactive against the sample species, and other antibodies\u2019 host species is desirable to minimize non-specific background.<\/span><\/p>\n<h5>Example 1: Indirect Sandwich ELISA with Human serum sample<\/h5>\n<table class=\"blog-tbl\"><caption>Table 1: Setting up a 3-step indirect sandwich ELISA using a combination of polyclonal and monoclonal antibodies<\/caption>\n<thead>\n<tr>\n<th>Antibody identity<\/th>\n<th>Clonality<\/th>\n<th>Specificity<\/th>\n<th>Host<\/th>\n<th>Min x<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Capture<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Monoclonal<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Antigen A<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Rat<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<\/tr>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Primary<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Polyclonal<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Antigen A<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Rabbit<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<\/tr>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Detection<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Polyclonal<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Rabbit<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Donkey<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Hu, Rat, Bov<\/span><\/p>\n<\/td>\n<\/tr>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Blocking<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<td>\u00a0<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">BSA<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/cross-adsorbed-secondary-antibodies\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about cross-reactivity<\/a><\/div>\n<\/div>\n<h4><span style=\"font-weight: 400;\">Orientation<\/span><\/h4>\n<p><span style=\"font-weight: 400;\">Although the antibody pairs may be interchangeable as the capture or primary antibody, the orientation of the antibody can impact the sensitivity and specificity of the assay. You may wish to compare how your antibodies perform in either role. We typically choose to use a min X antibody as the detection antibody.<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Another aspect of orientation to consider is which part or region of the antibody specificity is directed towards. Fc and F(ab\u2019)<sub>2<\/sub> specific antibodies are available and can be implemented if targeting of particular epitopes are required.\u00a0<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10496 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-10.png\" alt=\"\" width=\"642\" height=\"640\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-10.png 642w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-10-300x300.png 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-10-150x150.png 150w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Asset-10-45x45.png 45w\" sizes=\"(max-width: 642px) 100vw, 642px\" \/>\n<p>\u00a0<\/p>\n<figcaption><strong>Figure 3:<\/strong> Fc and F(ab\u2019)<sub>2<\/sub> specific antibodies can be used to \u201corient\u201d an assay towards a particular region of the partnered antibody in cases where particular epitopes may need to be preferentially bound in a particular order or direction.<\/figcaption>\n<\/figure>\n<h4><span style=\"font-weight: 400;\">Blocking<\/span><\/h4>\n<p><span style=\"font-weight: 400;\">After the capture antibody is applied, the plate is typically blocked using serum from the same host species as the conjugated detection antibody, or with\u00a0 <\/span><a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/001-000-162\"><span style=\"font-weight: 400;\">IgG and protease-free Bovine serum albumin (BSA)<\/span><\/a><span style=\"font-weight: 400;\">.<\/span><\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/elisa-guide-part-2\/\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about blocking for ELISA<\/a><\/div>\n<h4><span style=\"font-weight: 400;\">The sample<\/span><\/h4>\n<p><span style=\"font-weight: 400;\">The nature of the sample can add extra consideration to the assay. Matrix effects are the artifacts that are caused by the components of the sample, such as lipids, sugars and proteins, which can change the way an ELISA performs. Dilution of the sample can sometimes reduce the impact of these matrix effects and improve the signal-to-noise ratio.<\/span><\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/elisa-guide-part-1\/\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about ELISA<\/a><\/div>\n<hr \/>\n<h3>Detection of IgE from plasma using a direct sandwich ELISA<\/h3>\n<h5>Example 2: Human serum<\/h5>\n<table class=\"blog-tbl\"><caption>Table 2: A 2-Step direct sandwich ELISA set-up<\/caption>\n<thead>\n<tr>\n<th>Antibody<\/th>\n<th>Clonality<\/th>\n<th>Specificity<\/th>\n<th>Species<\/th>\n<th>Conjugate<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Capture<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Monoclonal<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Human IgE<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Mouse<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<\/tr>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Detection<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Monoclonal<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Human IgE<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Mouse<\/span><\/p>\n<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">Alkaline Phosphatase<\/span><\/p>\n<\/td>\n<\/tr>\n<tr>\n<td>\n<p><span style=\"font-weight: 400;\">Blocking<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<td>\u00a0<\/td>\n<td>\n<p><span style=\"font-weight: 400;\">BSA<\/span><\/p>\n<\/td>\n<td>\u00a0<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p><span style=\"font-weight: 400;\">Jackson ImmunoResearch Anti-Human IgE offers excellent sensitivity when used in a sandwich ELISA with its capture partner antibody.\u00a0<\/span><\/p>\n<p><span style=\"font-weight: 400;\">In this format, use our unconjugated Anti-Human IgE (<\/span><span style=\"font-weight: 400;\"> clone number 10A10 <\/span><span style=\"font-weight: 400;\"><a href=\"\/catalog\/products\/209-002-244\">209-002-244<\/a>) as your coating antibody before blocking with Bovine Serum Albumin (IgG-Free, Protease-Free <\/span><a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/001-000-161\"><span style=\"font-weight: 400;\">001-000-161<\/span><\/a><span style=\"font-weight: 400;\">). Following washing, incubate with your diluted sample.\u00a0<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10587\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-scaled.jpg\" alt=\"\" width=\"700\" height=\"700\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-scaled.jpg 2560w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-300x300.jpg 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-1024x1024.jpg 1024w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-150x150.jpg 150w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-1536x1536.jpg 1536w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-2048x2048.jpg 2048w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/2306_IgE_ELISA_graphic_JL_01ML2-45x45.jpg 45w\" sizes=\"(max-width: 700px) 100vw, 700px\" \/>\n<p>\u00a0<\/p>\n<figcaption><strong>Figure 4:<\/strong> Direct Sanwich ELISA. Unconjugated Anti-Human IgE ( clone number 10A10 <span style=\"font-weight: 400;\">209-002-244<\/span>) is used as the capture antibody and a conjugated Anti-Human IgE antibody, such as Alkaline Phosphatase Anti-Human IgE (209-052-241) is used as the detection antibody.<\/figcaption>\n<\/figure>\n<p><span style=\"font-weight: 400;\">For the detection step, use a conjugated Anti-Human IgE antibody, such as Alkaline Phosphatase Anti-Human IgE (<\/span><a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/209-052-241\"><span style=\"font-weight: 400;\">209-052-241<\/span><\/a><span style=\"font-weight: 400;\">) or Peroxidase Anti-Human IgE (<\/span><a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/209-032-241\"><span style=\"font-weight: 400;\">209-032-241<\/span><\/a><span style=\"font-weight: 400;\">), followed by the appropriate substrate or visualization technique.\u00a0<\/span><\/p>\n<p><span style=\"font-weight: 400;\">Figure 5 illustrates the application of the direct sandwich assay to validate the quantification of total IgE from patient samples using unconjugated Anti-Human IgE in partnership with an Alkaline Phosphatase conjugated detection antibody. Total IgE (kIU\/l) in patient samples used in this experiment were previously measured by the Roche Cobas Total IgE assay. These results are displayed on the x-axis of Figure 5.<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10449\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/serum-igEb.jpg\" alt=\"\" width=\"600\" height=\"528\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/serum-igEb.jpg 1440w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/serum-igEb-300x264.jpg 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/serum-igEb-1024x900.jpg 1024w\" sizes=\"(max-width: 600px) 100vw, 600px\" \/>\n<p>\u00a0<\/p>\n<figcaption><strong>Figure 5:<\/strong> Detection of total IgE from human serum and plasma samples by direct sandwich ELISA. Plate was coated with unconjugated Anti-Human IgE (clone 10A10 <a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/209-002-244\">209-002-244<\/a>) antibody at 100\u03bcl\/well at 2\u03bcg\/ml in coating buffer overnight at 4<sup>o<\/sup>C. The plate was blocked with 300\u03bcl\/well of 1% BSA (<a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/001-000-161\">001-000-161<\/a>) in Phosphate buffered saline, 0.05% Tween\u00ae (PBS\/T) for 1.5 hrs. Incubate with 100\u03bcl\/well of plasma\/serum samples diluted 1:80 in PBS\/T for 1hr. Detection antibody, Jackson ImmunoResearch Alkaline Phosphatase Anti-Human IgE (<a href=\"https:\/\/www.jacksonimmuno.com\/catalog\/products\/209-055-241\">209-055-241<\/a>), was diluted at 1:10,000 in PBS\/T and incubated at 100\u03bcl\/well for 1 hr. 100\u03bcl\/well of freshly prepared DEA substrate was incubated on the plate for 30 min and read at 405nm.<\/figcaption>\n<\/figure>\n<div class=\"btn-container bottom-btns\"><a class=\"btn-sq\" href=\"\/technical\/products\/groups\/anti-human-secondary-antibodies\/ige\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about Anti-Human IgE<\/a><br \/><a class=\"btn-sq\" href=\"\/catalog\/50\"><i class=\"fa-solid fa-magnifying-glass\"><\/i>Browse IgE antibodies<\/a><br \/><a class=\"btn-sq\" href=\"mailto:tech@jacksonimmuno.com\"><i class=\"fa-solid fa-message-lines\"><\/i>Speak to technical service about setting up an ELISA<\/a><\/div>\n<p><a href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/groups\/anti-human-secondary-antibodies\/ige\"><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10443 size-full\" style=\"width: 100%;\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/600x100-100.jpg\" alt=\"\" width=\"601\" height=\"101\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/600x100-100.jpg 601w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/600x100-100-300x50.jpg 300w\" sizes=\"(max-width: 601px) 100vw, 601px\" \/><\/a><\/p>\n<table class=\"table blogLinks\">\n<thead>\n<tr>\n<th class=\"span6\">Learn more:<\/th>\n<th class=\"span6\">Do more:<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/directandindirectwesternblotting\/\">Indirect and direct Western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"\/home\/exhibitions\">Exhibition Schedule<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/chemiluminescent-western-blotting\/\">Chemiluminescence western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/western-blotting-guide\/\">Western Blotting guide<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a class=\"row-title\" href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-admin\/post.php?post=3642&amp;action=edit\" aria-label=\"\u201cAn Introduction to Expansion Microscopy\u201d (Edit)\">An Introduction to Expansion Microscopy<\/a><\/td>\n<td class=\"span6\"><a href=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/immuno-techniques\/elisa-guide\/\">ELISA guide<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n<p><\/p>\n<!-- AddThis Advanced Settings generic via filter on the_content --><!-- AddThis Share Buttons generic via filter on the_content --><!-- AddThis Related Posts generic via filter on the_content -->","protected":false},"excerpt":{"rendered":"<p>Download PDF ELISA is a widely used technique for detecting and quantifying one or more specific proteins within a complex mixture. ELISAs are quick and easy to perform and can be adapted to detect almost any analyte, offering a highly sensitive technique that can be used to process large sample numbers. ELISAs can be performed [&hellip;]<!-- AddThis Advanced Settings generic via filter on get_the_excerpt --><!-- AddThis Share Buttons generic via filter on get_the_excerpt --><!-- AddThis Related Posts generic via filter on get_the_excerpt --><\/p>\n","protected":false},"author":3,"featured_media":10427,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"content-type":""},"categories":[19,3,16,20],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10415"}],"collection":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/comments?post=10415"}],"version-history":[{"count":54,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10415\/revisions"}],"predecessor-version":[{"id":10747,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/10415\/revisions\/10747"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media\/10427"}],"wp:attachment":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media?parent=10415"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/categories?post=10415"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/tags?post=10415"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}