Immunoassays, such as the ELISA, typically consist of two or more incubation steps, separated by wash steps. ELISA is most commonly performed in a 96-well plate, which is coated with bound antigen or antibody. Following a blocking step, the coated plate is first incubated with either a primary antibody or an antigen. The unbound (low-affinity) antigen-antibody complexes are then removed with several wash steps. Next, the plate is incubated with a secondary, enzyme-conjugated antibody, which binds to the high-affinity antibody-antigen complex attached to the plate. Again, the plate is washed several times to remove unbound antibody, and finally a reagent is added that reacts with the enzyme-conjugated antigen-antibody complex and produces a measurable signal.
Numerous substrates can be used to detect the final antigen-antibody complex. The substrates fall into three categories: photometric, chemiluminescent and fluorescent. To be precise, the term “ELISA” refers to assays in which a photometric substrate is used. Assays using a chemiluminescent substrate are referred to as Luminescent Immunoassay (LIA), and those using a fluorescent, conjugated secondary antibody are referred to as Fluorescent Immunoassay (FIA).
One of the key steps to focus on for optimizing ELISAs is washing. The washing steps are necessary to reduce background signal related to unbound, conjugated antibody and thereby increase the assay’s signal-to-noise ratio. Washing between steps ensures that only specific (high-affinity) binding events are maintained, to cause signal at the final step. Insufficient washing can result in variation and high background, and thus poor results. This article will provide you with tips on washing your ELISA plates using an automated plate washer, with the goal of avoiding high background and high variation (between plates and between replicate wells).
Wash parameters
Several parameters affect the effectiveness of wash steps. The first major parameter is wash volume. Automated washers dispense wash fluid from tips attached to a wash head. If you experience high background or variation, you should adjust the wash volume to be at least as high, or preferably higher, than the coating volume of the well. Too small a volume leaves part of the assay surface unwashed and strongly increases the background. Washing volume must be equal in all wells. ELISA plate manufacturers typically list their coating volumes in the kit’s brochure. A coating volume commonly used in the industry is 200 µl. If this is the case for your kit, the manufacturer might recommend using a wash volume of 300 µl, to clean the entire wall of the well. Wash volume varies according to the ELISA-reagent manufacturer’s instructions. In general, the higher the wash volume, the less excess antibody or antigen is left over after an incubation step.
Rule of thumb for cycles
The second major parameter affecting wash effectiveness is the number of wash cycles. In general, the greater the number of wash cycles, the lower the background. However, too many wash cycles can reduce signal strength, making it difficult to measure. A good rule of thumb is to repeat the wash step three times after each incubation step with antibody or antigen. However, the manufacturer of your coated ELISA plate should suggest a recommended number of wash cycles. In general, manufacturer-coated plates require fewer washes than plates coated by end users. For end-user-coated ELISA plates, it’s important to optimize the number of wash cycles.
Another way to manipulate wash volume and number of wash cycles is to overfill the well with wash buffer. Normally, an individual well of a 96-well plate can hold 330 to 460 µl. However, some automated washers can be programmed to dispense more than the maximum volume (e.g., 1 ml). That is possible because dispensing is performed while the aspiration function is continuously on. In other words, as the dispenser dispenses more liquid, the aspirator aspirates the liquid away from the well. This technique increases wash volume but does not cause overflow into other wells.
Aspiration
Several minor parameters influence the effectiveness of the wash step. These parameters include, for example, aspirate height and aspiration location, both of which can be adjusted to reduce background (residual volume) and variation.
The residual fluid is termed “dirty,” because it contains unbound antigen or antibody that can increase background signal. The smaller the residual volume, the less residual liquid, and therefore less interfering “dirty” liquid is transferred to next steps in the procedure.
Aspiration height can significantly affect residual volume; if the distance from washing tips to well bottom is even slightly too great, it will increase residual volume drastically. And if washing tips are pressed against the well bottom, the effect will be decreased aspiration efficiency and increased residual volume.
Two types of washing heads are used commonly in microplate washers: rigid and floating. A floating head contains washing tips that move freely up and down during washing, and rigid heads are fixed in place. Washing procedures with rigid heads are more complicated to optimize, because one needs to adjust aspiration height very carefully; height adjustment requires accuracy of about 0.1 mm. This can be time consuming if several different plate types are used. When floating heads are used, washing tips drop to the well bottom. Adjusting the height is not very important with floating heads, because the head can run so deep that it reaches the well bottom. Therefore, aspiration happens at the level of the well that gives the lowest possible residual volume.
Location of aspiration also plays an important role in residual volume; if only one aspiration point per well is used (which is common, because it is a little faster), that location should be optimized. The optimal location depends on the shape of the washing head, but it is almost never at the center of the well, even though this is the most common default location of all washers. Generally, the optimal location is somewhere between well center and well wall.
The shape of the well also affects residual volume. Microplates with a so-called "C-bottom"—a flat bottom with rounded corners—generally give remarkably lower residual volumes than totally flat-bottom plates with sharp corners.
Taken together, it’s clear the wash protocol always should be carefully examined and tested before the actual assay washes are performed.
Related Products from: Thermo Scientific