ELISA Technical Tips
Enzyme-linked immunosorbent assay (ELISA or EIA) is a plate-based assay used to measure the concentration of an analyte in a sample. The analyte may be a peptide, protein, antibody or hormone. Depending on the approach you choose, an antibody or antigen is immobilized on the plate’s surface. The sample is added to the plate followed by a detection reagent linked to an enzyme, such as HRP or AP. Finally, a substrate that reacts with the enzyme is added to generate a measurable signal.
Common ELISA Formats
There are many ELISA formats to choose from: direct, indirect, sandwich or competition. Always choose the simplest format to increase assay robustness. The “Sandwich” ELISA is most commonly used due to its robustness and sensitivity, but there are many other options. Another format may be more appropriate if you need a quick assay, if you only have one antibody or if you are experiencing cross-reactivity with the secondary antibody used for detection.
The first step in generating high quality data is to pay close attention to your pipetting skills. Pipetting accurately, consistently and quickly are very important. This prevents the wells from drying in-between washing steps and uneven reagent incubation. Electronic multichannel pipettes are a great tool for adding reagents to the plate, just make sure to use the correct tips and that the tips are firmly seated on the pipette. It is helpful to place a dark piece of paper underneath your plate to help visualize the wells while pipetting. The plate washing step is another common source of variability. When using a plate washer, make sure that it is primed properly and that the settings are appropriate for your plate. After the final wash is complete, don’t forget to tap the plate on a paper towel to remove residual liquid.
Antibodies are the key factor determining the sensitivity and specificity of your assay. Once the antibodies have been carefully selected, they should be titrated to maximize assay performance. Use a checkerboard titration to assess two reagents simultaneously to save time and reagents.
Whenever possible, confirm that your capture antibody is detecting the correct protein with a Western blot. Cross-validation of your antibody will give you confidence that you are measuring the correct analyte. If you don’t have the resources to cross-validate your antibody use a search engine such as CiteAb to see if a fellow scientist has generated the data you need. https://www.citeab.com/
High background can be troublesome since it decreases the sensitivity and specificity of your assay. Blocking your plate prevents nonspecific binding that decreases your signal-to-noise ratios. Unfortunately, the blocking reagent can also cause problems. The go-to buffer for blocking ELISA plates is typically 1-2% BSA with 0.05% Tween 20. Although this works well for most assays, signal may be suppressed and matrix effects may be observed with some samples. When BSA is not a good match for your assay, you can try Non-Fat Dry Milk, Casein, Fish Gelatin or other serum proteins. The process of finding the best buffer is often very time consuming and expensive. To help expedite this process, we have developed AdvanBlock™-EIA for those who have troublesome background or observe matrix effects with their samples. AdvanBlock™-EIA is a complex mix comprised of a proprietary blend of an artificially processed carbohydrate, a non-ionic detergent, a modified PEG, an anti-microbial agent and a processed and denatured mammalian protein component.
AdvanBlock™-EIA Blocking Solution
ELISABright Chemiluminescent Substrate
10X EIA Coating Buffer
HRP Conjugated Secondary Antibodies