{"id":4382,"date":"2024-09-11T00:06:33","date_gmt":"2024-09-11T05:06:33","guid":{"rendered":"https:\/\/www.bocsci.com\/blog\/?p=4382"},"modified":"2024-09-11T22:35:31","modified_gmt":"2024-09-12T03:35:31","slug":"elisa-protocols","status":"publish","type":"post","link":"https:\/\/www.bocsci.com\/blog\/elisa-protocols\/","title":{"rendered":"ELISA Protocols"},"content":{"rendered":"\n<p>These protocols are for general guidance only. For specific protocols, please refer to the product description page of the corresponding ELISA kit.<\/p>\n\n\n\n<h2><strong>Sandwich assay procedure<\/strong><strong><\/strong><\/h2>\n\n\n\n<ol type=\"1\">\n<li>An appropriate amount of trapping antibody (typically 100 \u03bcL) is added to each well of the 96-well plate at a concentration that follows product instructions or optimization results (typically 1-10 \u03bcg\/mL).&nbsp;Incubate at 4\u2103 overnight or at room temperature for 2 hours.<\/li>\n\n\n\n<li>Remove the trapping antibody solution from the well&nbsp;and flush the plate 3 times with the wash buffer.&nbsp;200 \u03bcL sealing buffer was added to each well and incubated at room temperature for 1-2 hours to block the unbound binding sites.<\/li>\n\n\n\n<li>Remove the sealing solution and rinse the plate&nbsp;3 times with the wash buffer.&nbsp;Add 100\u03bcL of test sample or standard product per well (diluted to the required concentration in advance) and incubate at room temperature for 1-2 hours or at 4\u2103 overnight.<\/li>\n\n\n\n<li>Remove the sample and standard solution and rinse the board 3-5 times with the wash buffer.&nbsp;Add 100 \u03bcL of enzyme-bound detection antibody per well (diluted at optimal concentration) and incubate at room temperature for 1-2 hours.&nbsp;<\/li>\n<\/ol>\n\n\n\n<p>5. Rinse the plate 3-5 times with a wash buffer to thoroughly remove unbound detection antibodies.&nbsp;Add 100 \u03bcL enzyme substrate solution to each well and react away from light (generally incubate at room temperature for 10-30 minutes, the specific time according to the substrate reaction rate).&nbsp;Observe color changes (usually the darker the color, the higher the antigen concentration).<\/p>\n\n\n\n<p>6. Add 50-100 \u03bcL stopping solution per well to stop the enzyme reaction.&nbsp;After termination of the reaction, the color will change from blue to yellow (TMB substrate for example).<\/p>\n\n\n\n<p>7. The light absorption value (OD value) of each hole is read using a microplate reader at 450 nm wavelength.&nbsp;The standard curve is drawn from the standard product, and the concentration of the target antigen is calculated according to the absorption value of the sample.<\/p>\n\n\n\n<p><strong>Chemicals used in sandwich assay<\/strong><\/p>\n\n\n\n<figure class=\"wp-block-table\"> \n<table>\n  <tr>\n    <td>classification<\/td>\n    <td>Name<\/td>\n    <td>CAS<\/td>\n  <\/tr>\n  <tr>\n    <td rowspan=\"4\">Phosphate    buffer (PBS)<\/td>\n    <td>Sodium    chloride<\/td>\n    <td>7647-14-5<\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/potassium-chloride-cas-7447-40-7-97879.html\">Potassium    chloride<\/a><\/td>\n    <td>7447-40-7<\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/monosodium-phosphate-cas-7558-80-7-180781.html\">Sodium    dihydrogen phosphate<\/a><\/td>\n    <td>7558-80-7<\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/disodium-hydrogenorthophosphate-cas-7558-79-4-98033.html\">Disodium    hydrogen phosphate<\/a><\/td>\n    <td>7558-79-4<\/td>\n  <\/tr>\n  <tr>\n    <td>Washing    buffer (PBS-T)<\/td>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/polysorbate-20-cas-9005-64-5-79252.html\">Tween-20<\/a><\/td>\n    <td>9005-64-5<\/td>\n  <\/tr>\n  <tr>\n    <td>Blocker<\/td>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/blood-serum-albumins-cas-9048-46-8-382204.html\">BSA(Bovine    serum albumin)<\/a><\/td>\n    <td>9048-46-8<\/td>\n  <\/tr>\n  <tr>\n    <td>Enzyme<\/td>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/horseradish-peroxidase-cas-9003-99-0-12686.html\">Horseradish    peroxidase<\/a><\/td>\n    <td>9003-99-0<\/td>\n  <\/tr>\n  <tr>\n    <td rowspan=\"5\">Substrate<\/td>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/3-3-5-5-tetramethylbenzidine-cas-54827-17-7-320701.html\">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine<\/a><\/td>\n    <td>54827-17-7 <\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/3-3-5-5-tetramethylbenzidine-dihydrochloride-cas-64285-73-0-324162.html\">3,3&#8242;,5,5&#8242;-Tetramethylbenzidine    dihydrochloride<\/a><\/td>\n    <td>64285-73-0 <\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/abts-chromophore-diammonium-salt-cas-30931-67-0-89872.html\">ABTS    Chromophore Diammonium Salt<\/a><\/td>\n    <td>30931-67-0 <\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/mtt-cas-298-93-1-11297.html\">MTT<\/a><\/td>\n    <td>298-93-1 <\/td>\n  <\/tr>\n  <tr>\n    <td><a href=\"https:\/\/www.bocsci.com\/product\/cspd-cas-142456-88-0-51725.html\">CSPD<\/a><\/td>\n    <td>142456-88-0 <\/td>\n  <\/tr>\n  <tr>\n    <td>Stop    solution<\/td>\n    <td>1M    H2SO4<\/td>\n    <td>7664-93-9<\/td>\n  <\/tr>\n<\/table>\n<\/figure>\n\n\n\n<h2><strong>Enzyme immunoassay<\/strong><strong>&nbsp;(<\/strong><strong>EIA<\/strong><strong>)<\/strong><strong>&nbsp;procedure<\/strong><strong><\/strong><\/h2>\n\n\n\n<ol type=\"1\">\n<li>The 96-well microtitration plate was removed, 100 \u00b5l was added to each well to capture the antibody, and the antibody was diluted to the appropriate concentration using the coated buffer.<\/li>\n<\/ol>\n\n\n\n<p>Incubate at 4\u2103 overnight or at room temperature for 2 hours.<\/p>\n\n\n\n<ul>\n<li>Wash the well&nbsp;three times with a wash buffer (PBST) of 350 \u00b5l each time.&nbsp;Shock well to ensure no residual liquid.<\/li>\n\n\n\n<li>Add 200 \u00b5l sealing buffer to each well.&nbsp;Incubate at room temperature for 1 hour to reduce nonspecific binding.&nbsp;Wash the microtitration plate 3 times to remove excess sealer.<\/li>\n\n\n\n<li>Add 100 \u00b5l sample or standard to each well and dilute the sample to the appropriate concentration.&nbsp;Incubate at room temperature for 2 hours.<\/li>\n\n\n\n<li>Wash the board 5 times with a wash buffer (PBST) of 350 \u00b5l each time.&nbsp;<\/li>\n\n\n\n<li>Add 100 \u00b5l to each well to detect the antibody and dilute the antibody to the appropriate concentration using a blocking buffer.&nbsp;Incubate at room temperature for 1 hour.<\/li>\n\n\n\n<li>Discard the solution. Repeat the wash procedure as in step 5.<\/li>\n\n\n\n<li>Add 100 \u00b5l substrate solution (TMB color developer) to each well.&nbsp;Incubate away from light at room temperature for 15-30 minutes until color develops.<\/li>\n\n\n\n<li>Add 50 \u00b5l stop solution (2N sulfuric acid) to each well.&nbsp;Incubate for a few minutes until all reactions are completely stopped.<\/li>\n\n\n\n<li>The optical density values of each well&nbsp;are read using an enzyme-labeler at a wavelength of 450 nm (or at a wavelength suitable for the substrate used).&nbsp;Calculate the concentration of antigen in the sample according to the standard curve.<\/li>\n<\/ul>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"aligncenter size-full\"><a href=\"https:\/\/www.bocsci.com\/blog\/wp-content\/uploads\/2024\/09\/Galactomannan-antigenemia-detection-in-the-Platelia-Aspergillus-EIA.jpg\"><img decoding=\"async\" loading=\"lazy\" width=\"588\" height=\"459\" src=\"https:\/\/www.bocsci.com\/blog\/wp-content\/uploads\/2024\/09\/Galactomannan-antigenemia-detection-in-the-Platelia-Aspergillus-EIA.jpg\" alt=\"Enzyme immunoassay schematic diagram\" class=\"wp-image-4385\" srcset=\"https:\/\/www.bocsci.com\/blog\/wp-content\/uploads\/2024\/09\/Galactomannan-antigenemia-detection-in-the-Platelia-Aspergillus-EIA.jpg 588w, https:\/\/www.bocsci.com\/blog\/wp-content\/uploads\/2024\/09\/Galactomannan-antigenemia-detection-in-the-Platelia-Aspergillus-EIA-300x234.jpg 300w\" sizes=\"(max-width: 588px) 100vw, 588px\" \/><\/a><figcaption class=\"wp-element-caption\">Galactomannan antigenemia detection in the Platelia\u00ae Aspergillus EIA. (Wheat, L. J.,&nbsp;2008)<\/figcaption><\/figure><\/div>\n\n\n<h2><strong>Phosphorylation assay procedure<\/strong><strong><\/strong><\/h2>\n\n\n\n<ol type=\"1\">\n<li>Dilute the capture antibody in coating buffer (usually carbonate-bicarbonate buffer, pH 9.6).&nbsp;Add 100 \u00b5L of the capture antibody solution to each well of the ELISA plate.&nbsp;Incubate the 96-well plate overnight at 4\u00b0C or for 2 hours at room temperature to allow the antibody to adsorb to the plate.<\/li>\n\n\n\n<li>Wash the wells three times with washing buffer to remove unbound antibodies.&nbsp;Block non-specific binding sites by adding 200 \u00b5L of blocking buffer to each well.&nbsp;Incubate the plate for 1 hour at room temperature.<\/li>\n\n\n\n<li>Remove the blocking buffer and wash the plate three times with washing buffer.&nbsp;Add 100 \u00b5L of cell lysates or purified protein samples to each well.&nbsp;Incubate the plate for 2 hours at room temperature or overnight at 4\u00b0C, allowing the target proteins to bind to the capture antibody.<\/li>\n\n\n\n<li>Wash the wells three times with washing buffer to remove unbound proteins.&nbsp;Add 100 \u00b5L of the specific anti-phosphorylation antibody to each well.&nbsp;Incubate the plate for 1 hour at room temperature.<\/li>\n\n\n\n<li>Wash the plate thoroughly with washing buffer to remove unbound primary antibodies.&nbsp;Add 100 \u00b5L of HRP-conjugated secondary antibody to each well.&nbsp;Incubate the plate for 1 hour at room temperature in the dark.<\/li>\n\n\n\n<li>Wash the plate again, ensuring thorough removal of any unbound secondary antibody.&nbsp;Add 100 \u00b5L of TMB substrate solution to each well.&nbsp;Incubate the plate at room temperature for 15-30 minutes, monitoring the development of the blue color indicating enzyme activity.<\/li>\n\n\n\n<li>Add 50 \u00b5L of stop solution to each well to halt the enzyme-substrate reaction. The addition of the acid will turn the color from blue to yellow.&nbsp;<\/li>\n\n\n\n<li>Measure the absorbance at 450 nm using a plate reader within 30 minutes of adding the stop solution.&nbsp;Perform data analysis comparing the absorbance values of sample wells to that of standard or control wells.<\/li>\n<\/ol>\n\n\n\n<h2><strong>Cell-based assay procedure<\/strong><strong><\/strong><\/h2>\n\n\n\n<h3><strong>1.&nbsp;<\/strong><strong>Preparation work<\/strong><strong><\/strong><\/h3>\n\n\n\n<p><strong>Reagents and consumables:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>The liquid nitrogen preserved cells must be resuscitated in advance and cultured to a suitable state.<\/p>\n\n\n\n<p>Gather all necessary reagents, including media, fixatives, blockers, primary and secondary antibodies, substrate solutions, and washing buffers.&nbsp;<\/p>\n\n\n\n<p>96-well plates, cell types suitable for culture conditions.<\/p>\n\n\n\n<p><strong>Cell culture:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Appropriate amount of specific cells were taken and cultured in culture bottles or petri dishes according to conventional cell culture methods.<\/p>\n\n\n\n<p>Cells were collected, cell density was counted, cell concentration was adjusted, and appropriate amount of cells were implanted into the 96-well plate. The number of cells per well should be consistent, usually adding 5,000 to 10,000 cells per well.<\/p>\n\n\n\n<p>The cells were incubated in an incubator at 37\u00b0C, 5% CO2, until the cells were overgrown with a moderate density (typically 70-90% confluent).<\/p>\n\n\n\n<h3><strong>2.&nbsp;<\/strong><strong>Fixation and blocking<\/strong><strong><\/strong><\/h3>\n\n\n\n<p><strong>Cell fixation:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Discard the medium and gently wash the cells with PBS per well. Add a fixative (e.g., 4% paraformaldehyde) to each hole and keep it fixed at room temperature or suitable temperature for 10-20 minutes.&nbsp;Wash with PBS several times to remove fixative. Care should be taken to prevent excessive washing resulting in cell shedding.<\/p>\n\n\n\n<p><strong>Cell penetration (if required):<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Add an appropriate amount of 0.1% Triton X-100 or other suitable penetrant to make the cell membrane semi-permeable so that the antibody can enter the cell interior.&nbsp;The osmotic treatment is usually carried out at room temperature for 5-10 minutes and washed again with PBS.<\/p>\n\n\n\n<p><strong>Block:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>A blocker (5% BSA or 10% normal serum is commonly selected) is added to each well for 1 hour at room temperature to reduce nonspecific binding.<\/p>\n\n\n\n<h3><strong>3.&nbsp;<\/strong><strong>Antibody incubation<\/strong><strong><\/strong><\/h3>\n\n\n\n<p><strong>Primary antibody incubation:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Select the specific primary antibody for the target protein and prepare it according to the concentration recommended in the instructions.&nbsp;An appropriate amount of primary antibody solution is added to each well and is usually left overnight at 4\u00b0C or incubated at room temperature for 1-2 hours.<\/p>\n\n\n\n<p><strong>Washing:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>The cells on the 96-well plate were washed multiple times with PBST (PBS + 0.05% Tween-20) or an appropriate washing buffer, usually for 3-5 minutes each time, repeated 3-5 times.<\/p>\n\n\n\n<p><strong>Secondary antibody incubation:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Select a suitable enzyme-labeled secondary antibody (usually HRP or AP labeled antibody) and formulate it according to the recommended concentration in the instructions.&nbsp;Add an appropriate amount of secondary antibody solution to each well and incubate at room temperature for 1-2 hours.<\/p>\n\n\n\n<p><strong>Wash again:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Repeat the above washing steps to remove non-specific binding after incubation of secondary antibody.<\/p>\n\n\n\n<h3><strong>4.&nbsp;<\/strong><strong>Substrate reaction and detection<\/strong><strong><\/strong><\/h3>\n\n\n\n<p><strong>Substrate solution:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Add appropriate substrate solution (such as TMB for HRP, PNPP for AP) to protect the incubation from light.&nbsp;The reaction time is usually 10-30 minutes until the color changes significantly.<\/p>\n\n\n\n<p><strong>Stopping the reaction:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>After the substrate reaction to a suitable degree, add a termination solution (such as 1M H2SO4) to immediately stop the color reaction.<\/p>\n\n\n\n<h3><strong>5.&nbsp;<\/strong><strong>Reading and analysis<\/strong><strong><\/strong><\/h3>\n\n\n\n<p><strong>Photometric measurement:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Using an ELISA reader, readings are usually taken at a suitable wavelength, such as 450nm.&nbsp;The optical density (OD value) per hole reflects the relative level of the target protein content.<\/p>\n\n\n\n<p><strong>Data analysis:<\/strong><strong><\/strong><\/p>\n\n\n\n<p>The standard curve is applied to the experimental sample and the OD value is converted to the protein concentration.&nbsp;Statistical analysis of repeated experimental data, such as calculating the mean value and standard error.<\/p>\n\n\n\n<p><strong>Reference<\/strong><strong><\/strong><\/p>\n\n\n\n<p>Wheat, L. J., <em>et al.<\/em>&nbsp;Diagnosis of invasive aspergillosis by galactomannan antigenemia detection using an enzyme immunoassay. <em>European Journal of Clinical Microbiology &amp; Infectious Diseases<\/em>. 2008,&nbsp;27, 245-251.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>These protocols are for general guidance only. For specific protocols, please refer to the product description page of the corresponding ELISA kit. Sandwich assay procedure 5. Rinse the plate 3-5 [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":4388,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[895],"tags":[],"_links":{"self":[{"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/posts\/4382"}],"collection":[{"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/comments?post=4382"}],"version-history":[{"count":3,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/posts\/4382\/revisions"}],"predecessor-version":[{"id":4394,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/posts\/4382\/revisions\/4394"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/media\/4388"}],"wp:attachment":[{"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/media?parent=4382"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/categories?post=4382"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.bocsci.com\/blog\/wp-json\/wp\/v2\/tags?post=4382"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}