{"id":8524,"date":"2023-03-27T15:59:07","date_gmt":"2023-03-27T15:59:07","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=8524"},"modified":"2023-03-27T16:03:22","modified_gmt":"2023-03-27T16:03:22","slug":"overview-of-elisa-protocol","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/overview-of-elisa-protocol\/","title":{"rendered":"Overview of ELISA Protocol"},"content":{"rendered":"<p>ELISA is a biochemical technique that utilizes antibodies and an <span id=\"urn:enhancement-8972ecdc-7075-433d-8d9f-cdd30e9fc5f8\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span>-triggered color change to detect either antigens, such as <span id=\"urn:enhancement-30c218db-a107-4bd9-ab58-bbb93f0ce693\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/proteins\">proteins<\/span>, hormones, and peptides, or antibodies in a provided sample. The &#8220;indirect&#8221; and &#8220;sandwich&#8221; methods of ELISA are capable of detecting antigens or antibodies at low concentrations. ELISAs come in various configurations, depending on their intended use. ELISA can be categorized into two primary types as a solid-phase method: competitive assays that employ either an <span id=\"urn:enhancement-e8c93f92-0e7e-4518-a5b4-b081ac8d1205\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span>&#8211;<span id=\"urn:enhancement-a3d3b618-c038-49a8-96ba-a7c4ed0b5c41\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> conjugate or an antibody-<span id=\"urn:enhancement-b0efb489-4d9c-4710-bd4a-101495e393d6\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> conjugate, and non-competitive assays that use a double antibody &#8220;sandwich&#8221; technique where the second antibody has an indicator <span id=\"urn:enhancement-3dd595c5-1c2a-4208-be30-455256140a99\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> linked to it.<\/p>\n<p>The most important step in the ELISA assay is to directly or indirectly identify the <span id=\"urn:enhancement-34a9a7a2-62a8-4261-b69b-9045d3f79527\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> by attaching or immobilizing either the <span id=\"urn:enhancement-9da5e2a9-1813-4dee-a644-88cf13596e3d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or the <span id=\"urn:enhancement-ffe211c1-8d55-481e-8f6e-7ad1dd400bca\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span>-specific capture antibody onto the surface of the well. To achieve sensitive and reliable measurements, a &#8220;capture&#8221; antibody can selectively extract the <span id=\"urn:enhancement-75bb1261-b486-4d24-b72e-458d211deed9\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> from a mixture of antigens in the sample. This results in the <span id=\"urn:enhancement-25278e4b-c293-4b4c-a42f-64bac3405144\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> being &#8220;sandwiched&#8221; between the capture antibody and the detection antibody. In situations where the <span id=\"urn:enhancement-ef2f7fef-0c16-4789-b4c9-774dbe18cd2f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> to be measured is small or has only one <span id=\"urn:enhancement-6c721d74-13f4-4278-9fc6-2175ae22fb35\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/epitopes\">epitope<\/span> for antibody binding, a competitive approach is employed. In this approach, either the <span id=\"urn:enhancement-d2ed7c46-fd48-48d8-802f-a59a08d8b58a\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> is labeled and competes for the formation of the unlabeled <span id=\"urn:enhancement-b6e1c017-98c2-47f0-b77b-b1a8a32be59c\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span>-antibody complex or the antibody is labeled and competes for the <span id=\"urn:enhancement-f960ce2f-84d7-4e5a-8e3e-3769a4d10c29\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> bound to the sample. Each of these modified ELISA techniques can be used for both qualitative and quantitative purposes.<\/p>\n<p>&nbsp;<\/p>\n<p>The ELISA protocol involves several steps, which are outlined below:<\/p>\n<ul>\n<li>\n<h3><strong>Coating: <\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>Coating is a critical step in which an <span id=\"urn:enhancement-58460acf-d680-487e-9684-2be717a8f595\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or capture antibody is immobilized onto a microtiter plate to create a solid-phase matrix. The <span id=\"urn:enhancement-a876c5f1-05b9-4fc3-b4ba-61ffd55298cc\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or antibody is typically coated onto the wells of the microtiter plate by incubating the plate with a solution containing the <span id=\"urn:enhancement-a9e33848-3cd4-42bf-b8f8-4191f4030fd2\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or antibody. The choice of <span id=\"urn:enhancement-8ffec91e-3c5d-4902-8a9e-8efaf8f2609b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or antibody to be coated onto the plate depends on the specific application and the target molecule to be detected. Proper optimization of the coating step, including the concentration and <span id=\"urn:enhancement-11bc11e7-9c10-417c-84e1-d1d3b040bf3e\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/incubation\">incubation<\/span> time of the <span id=\"urn:enhancement-9c1b5061-c909-45b0-b6dd-64b23c760091\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or antibody, is essential to ensure the success of the assay.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-8526\" src=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/03\/3.2.png\" alt=\"\" width=\"400\" height=\"371\" \/><\/p>\n<p>&nbsp;<\/p>\n<ul>\n<li>\n<h3><strong>Blocking: <\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>Blocking is a step where non-specific <span id=\"urn:enhancement-bdd65966-f60a-4923-bb47-e85927dd2deb\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/binding-sites\">binding sites<\/span> on the microtiter plate are blocked to prevent non-specific binding of antibodies or other <span id=\"urn:enhancement-4176494d-a24c-4e96-8701-31bfe61d4033\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/proteins\">proteins<\/span> to the plate. Blocking is essential to reduce background noise and improve the specificity of the assay. Blocking is typically done by adding a protein such as bovine <span id=\"urn:enhancement-2543952d-e546-4d36-a42e-405a82842087\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/serum\">serum<\/span> albumin (BSA), <span id=\"urn:enhancement-58db641a-d2c9-4736-9b5d-9f29ebee0526\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/casein\">casein<\/span>, or milk to the wells after the plate has been coated with the primary antibody or <span id=\"urn:enhancement-bc5a7301-a55a-458a-b82c-3f80c4e6aa46\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span>. The blocking protein binds to any remaining unoccupied sites on the plate, reducing the likelihood of non-specific binding of antibodies or other <span id=\"urn:enhancement-df64faec-76d7-47d1-8a7f-ce929c22578a\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/proteins\">proteins<\/span> to the plate.<\/p>\n<p>The plate is then incubated to allow the blocking protein to bind to the plate, and any excess blocking protein is removed by washing the plate with a buffer solution. After blocking, the primary antibody or <span id=\"urn:enhancement-27662e2c-97df-4dc2-b8be-68554d660318\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> can be added to the wells.<\/p>\n<ul>\n<li>\n<h3><strong>Sample and standard addition: <\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>The standards are typically a set of known concentrations of the <span id=\"urn:enhancement-528fb08a-d520-4012-bec8-1f75fc4ce176\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> that are used to generate a calibration curve, which is used to quantify the amount of the <span id=\"urn:enhancement-0705c4bc-e0a3-49d3-9544-bb60d2768775\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> in the unknown samples. The standards are added in known concentrations in separate wells of the plate, whereas the unknown samples are added in duplicate or triplicate wells.<\/p>\n<ul>\n<li>\n<h3><strong>Primary antibody incubation:<\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>The primary antibody <span id=\"urn:enhancement-83f1b459-214d-4c55-a453-89e570c0ded1\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/incubation\">incubation<\/span> step involves the addition of a specific antibody that recognizes and binds to the <span id=\"urn:enhancement-b4425fa0-f0b6-493f-8242-0b02dfd7d630\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> of interest that has been coated onto the microtiter plate. The primary antibody is typically raised in a specific species, such as mouse or rabbit, and may be labeled or unlabeled. If the primary antibody is unlabeled, a <span id=\"urn:enhancement-41f2c1af-434b-411e-8bfe-e302984c3a3c\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> <span id=\"urn:enhancement-0adeb4b8-1750-4d71-b80f-dd4dae554ad4\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/conjugated\">conjugated<\/span> to an <span id=\"urn:enhancement-ba886ca7-b502-4ec2-b1e1-a9ee72caf718\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> such as horseradish peroxidase (HRP) or <span id=\"urn:enhancement-be99b544-9112-41c0-bbc6-4e8a07f79282\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/alkaline-phosphatase\">alkaline phosphatase<\/span> (AP) is added in a later step to generate a measurable signal.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-8525\" src=\"https:\/\/www.mybiosource.com\/learn\/wp-content\/uploads\/2023\/03\/3.1.jpg\" alt=\"\" width=\"352\" height=\"385\" \/><\/p>\n<p>&nbsp;<\/p>\n<ul>\n<li>\n<h3><strong>Secondary antibody incubation:<\/strong><\/h3>\n<\/li>\n<\/ul>\n<p><span id=\"urn:enhancement-31121a99-1915-4037-baf9-5c9152b3d4b3\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">Secondary antibody<\/span> <span id=\"urn:enhancement-42e77b1e-f7ff-4b49-a79d-ffec22fb5a53\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/incubation\">incubation<\/span> is a crucial step in the ELISA assay, where a <span id=\"urn:enhancement-3b1e7c83-902f-4055-a59e-038a82d64e01\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> is added to the microtiter plate to bind specifically to the primary antibody that was previously bound to the <span id=\"urn:enhancement-15f31966-3edf-44b8-aa51-6cb0b747d301\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> or antibody coated on the plate. The primary antibody-<span id=\"urn:enhancement-781cf046-4da1-4bb8-a54d-e10421cc9d8d\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> complex acts as a bridge to link the <span id=\"urn:enhancement-c40ce4ae-3a29-462c-b1f7-3454971b8283\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> to the microtiter plate, allowing the <span id=\"urn:enhancement-b6a3b280-4434-4d40-b8be-f9458eb2a6ff\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> to bind to the substrate and generate a measurable signal. The signal generated by the <span id=\"urn:enhancement-70e737fc-fb24-4ab0-897b-32c62d229b1b\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span>&#8211;<span id=\"urn:enhancement-757e72d7-8b2f-432b-98c5-88db6572bffa\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/conjugated\">conjugated<\/span> <span id=\"urn:enhancement-24df151a-1e39-4147-9cd2-83ffc7e65f9c\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> is proportional to the amount of the primary antibody or <span id=\"urn:enhancement-cedea937-18de-49a2-ad96-9443b3d3df7f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> present in the sample.<\/p>\n<ul>\n<li>\n<h3><strong>Substrate addition:<\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>In ELISA, a substrate is added to the microtiter plate after the <span id=\"urn:enhancement-f36b3cf0-bf4d-4b60-bf53-3c259d876cce\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/incubation\">incubation<\/span> of the <span id=\"urn:enhancement-cda15e74-4689-4618-98f3-1f19ef5f2646\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> to generate a measurable signal. The substrate reacts with the <span id=\"urn:enhancement-f824af03-3c06-414e-8e09-487844c6dccd\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span>&#8211;<span id=\"urn:enhancement-5b4c9c8a-814c-4d80-9af7-4f870cdff47d\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/conjugated\">conjugated<\/span> to the <span id=\"urn:enhancement-980c203e-62f4-4717-aed0-49b0aa523146\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span> and produces a detectable signal that can be quantified using a <span id=\"urn:enhancement-afa8b24b-1a1c-447f-b462-d1b3f5f26155\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/microplate-readers\">microplate reader<\/span>. The choice of substrate depends on the type of <span id=\"urn:enhancement-2190a1d3-7805-4708-a693-167eeeda57ee\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> used, and the most commonly used substrates are chromogenic or chemiluminescent. Chromogenic substrates produce a colored reaction product that can be read at a specific wavelength, whereas chemiluminescent substrates produce light, which can be measured by a luminometer. The reaction time and substrate concentration should be optimized to generate an adequate signal without causing background noise.<\/p>\n<ul>\n<li>\n<h3><strong>Stop solution addition:<\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>A stop solution is added to the microtiter plate to stop the <span id=\"urn:enhancement-c3f3726c-aaee-41e6-a4e5-e13d3fb49864\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzymatic-reaction\">enzymatic reaction<\/span> between the substrate and the <span id=\"urn:enhancement-35f8cd5a-26fe-4874-97ef-582a8f3be7ba\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span>&#8211;<span id=\"urn:enhancement-723186ca-d4a1-40cf-9d85-326c209644f4\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/conjugated\">conjugated<\/span> <span id=\"urn:enhancement-a2ebca7b-f052-47fa-af0a-360c8081d59c\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/secondary-antibody\">secondary antibody<\/span>. The stop solution changes the pH of the reaction mixture, which causes the <span id=\"urn:enhancement-a8cc5ea9-c485-488e-93c8-a26f1ac8b894\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzymatic-reaction\">enzymatic reaction<\/span> to stop and prevents further color development or luminescence. A commonly used stop solution is sulfuric acid, which is added to the wells after the substrate <span id=\"urn:enhancement-ba374a8c-4d0b-4b18-a9e1-069c0ce7227c\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/incubation\">incubation<\/span> step. The addition of the stop solution is essential to ensure that the reaction does not continue and that the signal generated is stable and reproducible. After adding the stop solution, the absorbance or luminescence of each well is measured using a <span id=\"urn:enhancement-a343a3b2-a4f0-4895-bc13-86edbdfb2a8a\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/microplate-readers\">microplate reader<\/span>.<\/p>\n<ul>\n<li>\n<h3><strong>Signal detection:<\/strong><\/h3>\n<\/li>\n<\/ul>\n<p>The signal is generated when the substrate is added to the microtiter plate and reacts with the <span id=\"urn:enhancement-85e93d97-2793-4539-baf7-b9e7d4965fd4\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/enzyme\">enzyme<\/span> to produce a colorimetric or <span id=\"urn:enhancement-a164d566-bb74-4c58-9c4e-cba31bd7f174\" class=\"textannotation disambiguated wl-thing\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/chemiluminescent\">chemiluminescent<\/span> signal, depending on the substrate used. The signal generated is proportional to the amount of the primary antibody or <span id=\"urn:enhancement-f8246e6b-3517-4960-b5d5-05064f69bb2f\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> present in the sample. The results are analyzed using software or other statistical methods to determine the concentration of the primary antibody or <span id=\"urn:enhancement-026c467f-9d78-4e2c-8fb3-3e26d706c633\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> in the sample.<\/p>\n<h3><span style=\"text-decoration: underline;\"><strong>Conclusion<\/strong><\/span><\/h3>\n<p>Each stage of the ELISA will influence the final result and therefore great care must be taken to optimize and then standardize the method. The most important influencing factors are <span id=\"urn:enhancement-69fe26a6-9f9d-4c2c-bce1-90092c3f7250\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> coating, choice of plates, choice of blocking agent, and choice of secondary antibodies and detection system. Each of these factors will vary with each <span id=\"urn:enhancement-2e4fac2d-6f9b-41bc-a372-cb850ddeed3e\" class=\"textannotation disambiguated wl-creative-work\" itemid=\"https:\/\/data.wordlift.io\/wl1503301\/entity\/antigen\">antigen<\/span> being used.<\/p>\n<p>Overall, the ELISA protocol is highly sensitive and has numerous applications in research, clinical diagnosis, and drug development.<\/p>\n<p><strong>References<\/strong><\/p>\n<ol>\n<li>(PDF) Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay (researchgate.net)<\/li>\n<li>Reen, D. J. (n.d.). Enzyme-Linked Immunosorbent Assay (ELISA). Basic Protein and Peptide Protocols, 461\u2013466. doi:10.1385\/0-89603-268-x:461<\/li>\n<li>https:\/\/link.springer.com\/protocol\/10.1385\/1-59259-321-6:243<\/li>\n<li>Conceptual view of the ELISA assay. Streptavidin-coated (blue ovals)&#8230; | Download Scientific Diagram (researchgate.net)<\/li>\n<\/ol>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>ELISA is a biochemical technique that utilizes antibodies and an enzyme-triggered color change to detect either antigens, such as proteins, hormones, and peptides, or antibodies in a provided sample. The &#8220;indirect&#8221; and &#8220;sandwich&#8221; methods of ELISA are capable of detecting antigens or antibodies at low concentrations. ELISAs come in various configurations, depending on their intended [&hellip;]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-8524","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/8524","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=8524"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/8524\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=8524"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}