Troubleshooting ELISAs

Troubleshooting ELISAs

<div class="level1"> The enzyme-linked immunosorbent assay (ELISA) is a powerful technique for identifying and quantifying proteins. The assay is less complex than other assays, however problems can still arise when performing ELISAs. These problems can lead to little or no signal, or a high background that obscures the true signal. This troubleshooting guide lists common issues associated with ELISAs and solutions to these issues. </div> <h2 id="poor_signal" class="sectionedit2">Poor signal</h2> <div class="level2"> Low signal can occur for a variety of reasons preventing detection of the target protein. </div> <h3 id="not_enough_target_protein_in_the_well" class="sectionedit3">Not enough target protein in the well</h3> <div class="level3"> A threshold of detection must be reached before the true signal can be detected above background. Sufficient target protein must be used to achieve this threshold. To increase target protein amounts: <ul> <li class="level1"> <div class="li">Concentrate samples prior to adsorption to the plate.</div></li> <li class="level1"> <div class="li">Use a<span class="Apple-converted-space"> </span><a class="urlextern" title="http://www.advansta.com/blog/sandwich-lab/" href="https://advansta.com/sandwich-lab/" target="_blank" rel="nofollow">sandwich ELISA</a><span class="Apple-converted-space"> </span>to capture and bind the target protein to the well.</div></li> <li class="level1"> <div class="li">Use an<span class="Apple-converted-space"> </span><a class="wikilink1" title="elisas" href="https://advansta.com/elisa-personality-direct-indirect/" target="_blank">indirect detection method</a><span class="Apple-converted-space"> </span>to enhance the signal.</div></li> <li class="level1"> <div class="li">If the target protein/peptide isn’t absorbing well to the plate either:</div> <ul> <li class="level2"> <div class="li">Conjugate the antigen to a carrier protein before coating the plate</div></li> <li class="level2"> <div class="li">Biotinylate the protein and use streptavidin to adsorb the protein to the plate</div></li> </ul> </li> <li class="level1"> <div class="li">Use a positive control.</div> <ul> <li class="level2"> <div class="li">Titrate the positive control to determine the limit of detection of the assay.</div></li> </ul> </li> </ul> </div> <h3 id="signal_too_low" class="sectionedit4">Signal too low</h3> <div class="level3"> Low signal can arise from the inefficient detection of the target protein. To increase the signal: <ul> <li class="level1"> <div class="li">Use highly specific antibodies.</div></li> <li class="level1"> <div class="li">Use an indirect detection method to enhance the signal.</div></li> </ul> </div> <h3 id="incorrect_orientation_of_the_target_protein" class="sectionedit5">Incorrect orientation of the target protein</h3> <div class="level3"> When using an ELISA to detect antibodies, the antibodies can orient improperly on the plate such that the binding site is no longer accessible to the detecting antibody. To properly orient antibodies, first coat the wells with a protein that binds to the Fc region of the antibody (Protein A or G). </div> <h3 id="capture_primary_or_detecting_antibody_concentrations_too_low" class="sectionedit6">Capture, primary or detecting antibody concentrations too low</h3> <div class="level3"> <a class="wikilink1" title="dot_blot" href="https://advansta.com/wikis/protocol-dot-blot-checkerboard-titration-of-antibodies-2/">Titrate all antibodies</a><span class="Apple-converted-space"> </span>used in the assay to determine the optimum working concentrations. </div> <h3 id="wells_dried_out" class="sectionedit7">Wells dried out</h3> <div class="level3"> Signal can be lost if wells dry out during incubations. Use sealing film or tape to cover wells. </div> <h3 id="poor_substrate" class="sectionedit8">Poor substrate</h3> <div class="level3"> Poor signal can occur if the substrate is not handled or stored correctly. <ul> <li class="level1"> <div class="li">Make sure the substrate is not expired and has been stored correctly (some kit components require different storage conditions – separate components and store individually).</div></li> <li class="level1"> <div class="li">Incubate the substrate in the dark if required.</div></li> <li class="level1"> <div class="li">Do not use wash buffers containing sodium azide. Sodium azide can interfere with some substrates.</div></li> </ul> </div> <h3 id="lab_temperature_too_low" class="sectionedit9">Lab temperature too low</h3> <div class="level3"> ELISAs are dependent on temperature and the experiment should be performed using the recommended temperature conditions. For most assays this is room temperature (25°C). <ul> <li class="level1"> <div class="li">Do not run the experiment near air conditioning vents or cold windows.</div></li> <li class="level1"> <div class="li">Make sure plates and reagents are at the correct temperature before use.</div></li> </ul> </div> <h3 id="washes_too_intense" class="sectionedit10">Washes too intense</h3> <div class="level3"> Overwashing can disrupt antibody/antigen interactions or remove the antigen from the plate. <ul> <li class="level1"> <div class="li">Optimize the wash buffer for each ELISA.</div></li> <li class="level1"> <div class="li">Shorten wash cycles by decreasing wash time or number of washes.</div></li> <li class="level1"> <div class="li">If using a plate washer, check for microbial contamination in the lines, which can cause a decrease in signal.</div></li> <li class="level1"> <div class="li">Prime the plate washer with wash solution to prevent contamination from previous user’s solutions.</div></li> </ul> </div> <h3 id="incubations_too_short" class="sectionedit11">Incubations too short</h3> <div class="level3"> Short incubations can interfere with formation of antigen-antibody complexes and inhibit color development. <ul> <li class="level1"> <div class="li">Increase incubation time with antibodies to allow complex formation.</div></li> <li class="level1"> <div class="li">Optimize substrate incubation time.</div></li> </ul> </div> <h2 id="high_background_false_positives" class="sectionedit12">High background/False Positives</h2> <div class="level2"> High background results in a low signal-to-noise ratio and an inability to properly detect true signal. Several factors can contribute to a high background. </div> <h3 id="matrix_effect" class="sectionedit13">Matrix effect</h3> <div class="level3"> Matrix effects occur when the composition of the sample leads to high background or false positives. Certain samples, such as those from plasma and serum, are more prone to matrix effects. To <a href="https://advansta.com/beware-of-matrix-effects-in-your-elisa-assay/" target="_blank">eliminate or test for matrix effects</a>: <ul> <li class="level1"> <div class="li">Dilute samples using the same diluent as the standard curve.</div></li> <li class="level1"> <div class="li">Use a range of concentrations of the sample.</div></li> </ul> </div> <h3 id="cross-reactivity" class="sectionedit14">Cross-reactivity</h3> <div class="level3"> Cross-reactivity occurs when the antibody recognizes another protein in the sample that has a high homology to the target protein. To avoid cross-reactivity: <ul> <li class="level1"> <div class="li">Use the most specific antibody available.</div></li> <li class="level1"> <div class="li">Use a more specific ELISA, such as a sandwich ELISA.</div></li> <li class="level1"> <div class="li">Try an antibody that recognizes a different epitope.</div></li> </ul> </div> <h3 id="non-specific_binding" class="sectionedit15">Non-specific binding</h3> <div class="level3"> Non-specific binding refers to binding of the assay antibodies in a manner not related to the specificity of the antibody. Non-specific binding can occur when antibodies bind to the wells or to a component of the blocking or wash buffers. To prevent non-specific binding: <ul> <li class="level1"> <div class="li">Make sure wells are completely blocked by incubating with an appropriate blocking buffer for the specified time.</div></li> <li class="level1"> <div class="li">Optimize the blocking and washing buffers to make sure they are compatible with the antibodies.</div></li> <li class="level1"> <div class="li">Increase the salt concentration or add detergent to the washing buffers.</div></li> </ul> </div> <h3 id="too_much_wrong_antibody_is_used" class="sectionedit16">Too much/wrong antibody is used</h3> <div class="level3"> Too much antibody or use of the wrong secondary antibody in an indirect ELISA can lead to overall high background. <ul> <li class="level1"> <div class="li"><a class="wikilink1" title="dot_blot" href="https://advansta.com/wikis/protocol-dot-blot-checkerboard-titration-of-antibodies-2/" target="_blank">Titrate all antibodies</a><span class="Apple-converted-space"> </span>to determine the optimum working concentrations.</div></li> <li class="level1"> <div class="li">When performing an indirect sandwich ELISA, make sure the detection antibody does not recognize the capture antibody.</div> <ul> <li class="level2"> <div class="li">Use highly-cross adsorbed secondary antibodies</div></li> <li class="level2"> <div class="li">Use capture and primary antibodies isolated from different species</div></li> </ul> </li> </ul> </div> <h3 id="cross_contamination_of_wells" class="sectionedit17">Cross contamination of wells</h3> <div class="level3"> Splashing between wells can cause mixing of samples between wells. Use sealing film or tape to cover all wells during incubations. </div> <h3 id="poor_substrate1" class="sectionedit18">Poor substrate</h3> <div class="level3"> Poor substrate or improper use of substrate can lead to high background. <ul> <li class="level1"> <div class="li">Do not use expired substrate.</div></li> <li class="level1"> <div class="li">Store substrate correctly (some kit components require different storage conditions – separate components and store individually).</div></li> <li class="level1"> <div class="li">Make sure the substrate is colorless before use. Deteriorated substrate will have a tinge of color.</div></li> <li class="level1"> <div class="li">Dilute substrate correctly.</div></li> <li class="level1"> <div class="li">Optimize the incubation time with substrate.</div></li> <li class="level1"> <div class="li">If required, add stop solution immediately when reaction reaches desired color.</div></li> <li class="level1"> <div class="li">Add stop solution efficiently to each well.</div></li> </ul> </div> <h3 id="lab_temperature_too_high" class="sectionedit19">Lab temperature too high</h3> <div class="level3"> Increased temperatures can contribute to high background. To decease effects due to high temperatures: <ul> <li class="level1"> <div class="li">Run assays at the recommended temperature.</div></li> <li class="level1"> <div class="li">Do not run assays near heaters or vents or in direct sunlight.</div></li> </ul> </div> <h3 id="inefficient_washing" class="sectionedit20">Inefficient washing</h3> <div class="level3"> Inefficient washing can lead to non-specific binding. To improve washing conditions: <ul> <li class="level1"> <div class="li">Change the salt concentration.</div></li> <li class="level1"> <div class="li">Include blocking reagent in wash buffer.</div></li> <li class="level1"> <div class="li">Include detergent in wash buffer.</div></li> <li class="level1"> <div class="li">Prime plate washers with correct wash buffer.</div></li> <li class="level1"> <div class="li">Increase wash time and number of washes.</div></li> </ul> </div> <h3 id="incubations_too_long" class="sectionedit21">Incubations too long</h3> <div class="level3"> Excessive incubation times can increase background. Optimize all antibody and substrate incubation times. </div> <h3 id="contaminated_reagents" class="sectionedit22">Contaminated reagents</h3> <div class="level3"> Microbial contamination of reagents and equipment can lead to high background. <ul> <li class="level1"> <div class="li">Make sure water is not contaminated.</div></li> <li class="level1"> <div class="li">Prepare blocking and wash buffers fresh.</div></li> <li class="level1"> <div class="li">If using an automatic washer, clean lines regularly to prevent contamination.</div></li> </ul> </div> <h3 id="poor_standard_curve" class="sectionedit23">Poor standard curve</h3> <div class="level3"> A poor <a href="https://advansta.com/dont-make-a-substandard-elisa-standard-curve/" target="_blank">standard curve</a> will lead to inaccurate quantification of samples. To prepare high-quality standard curves: <ul> <li class="level1"> <div class="li">Accurately prepare the stock standard.</div> <ul> <li class="level2"> <div class="li">Spin vial of unconstituted standard to collect everything on the bottom of the tube.</div></li> <li class="level2"> <div class="li">Reconstitute according to the manufacturer’s instructions.</div></li> <li class="level2"> <div class="li">Mix standard well.</div></li> <li class="level2"> <div class="li">Aliquot standard if freezing and thawing.</div></li> </ul> </li> <li class="level1"> <div class="li">Make dilutions carefully when setting up a standard curve.</div> <ul> <li class="level2"> <div class="li">Pipet samples carefully.</div></li> <li class="level2"> <div class="li">Mix samples thoroughly.</div></li> <li class="level2"> <div class="li">Use a fresh pipet tip between samples.</div></li> <li class="level2"> <div class="li">Use diluent compatible with assay.</div></li> <li class="level2"> <div class="li">Use same diluent for standard curve and when diluting samples.</div></li> </ul> </li> <li class="level1"> <div class="li">Use a standard curve that covers a range of concentrations appropriate for the unknown samples.</div></li> </ul> </div> <h2 id="erroneous_readings" class="sectionedit24">Erroneous readings</h2> <div class="level2"> To prevent plate reading errors: <ul> <li class="level1"> <div class="li">Avoid generating bubbles when pipetting samples. Bubbles in wells will affect readings.</div></li> <li class="level1"> <div class="li">If the substrate requires a stop solution, read the plate immediately after addition of stop solution to all wells.</div></li> </ul> Photo courtesy of <a href="https://www.flickr.com/photos/biologycourses/7152514131/in/photolist-bU3vga-bF8Q1Q-nqnnUz-AkU9fJ-nvxyTc-xiPctL-s6mXFi-pT4tbc-5FWRLD-8YHTGy-8c6pzo-wDoKfq-xB1WJT-wDoFKj-xy5YT7-xiPegW-xzAT4S-xiP49Q-wDxUJ6-xiNzyu-xiN4SW-xiMRWb-9DZkSp-6pDCkF-7dCnpr-qovvFB-dSYxnk-8VF8ga-8q2j1o-qvH9Yx-bKEoDr-ad9y35-aEnJpu-8VJdc9-8iTAKW-qCcAvH-qnqL6z-8iP9Wa-anhMky-c9RjE-9Xh1cb-3rudK1-eZFHL6-qiJ6HY-6A15Q1-u4G8o-adFgDy-8Mnn6y-8MoGA1-8LxCPz" target="_blank">Viven Rolf</a>e. </div>