Key points of ELISA experiment: washing is the key

One of the important points of optimizing ELISA is washing. The washing step reduces the background signal caused by unbound antibody, thereby increasing the signal-to-noise ratio of the analysis. The washing between each step ensures that only specific binding events are retained, generating a signal in the last step. Insufficient washing can lead to differences and high background, which can lead to undesirable results. This article will introduce you some techniques for washing ELISA plates using an automatic plate washer.

Immunoassays, such as ELISA, generally include two or more incubation steps, separated by washing. ELISA is usually carried out in 96-well plates, which are coated with bound antigens or antibodies. After the blocking step, the coated plate is first incubated with the primary antibody or antigen. Subsequent washing steps remove unbound (low affinity) antigen-antibody complexes. Next, the plate is incubated with the secondary antibody. This enzyme-containing antibody binds to high-affinity antigen-antibody complexes. After that, it was another round of washing. Finally, the substrate is added and the signal is detected.

We can use a variety of substrates to detect the final antigen-antibody complex. Substrates are divided into three categories: natural luminescence, chemiluminescence and fluorescence. To be precise, the term ELISA refers to analysis using naturally luminescent substrates. The analysis using chemiluminescent substrates is called Luminescent Immunoassay (LIA), and the analysis using fluorescent secondary antibodies is called Fluorescent Immunoassay (FIA).

One of the important points of optimizing ELISA is washing. The washing step reduces the background signal caused by unbound antibody, thereby increasing the signal-to-noise ratio of the analysis. The washing between each step ensures that only specific binding events are retained, generating a signal in the last step. Insufficient washing can lead to differences and high background, which can lead to undesirable results. This article will introduce you some techniques for washing ELISA plates using an automatic plate washer.

Washing parameters

Some parameters will affect the effectiveness of the washing step. The first major parameter is the amount of washing. The automatic washing machine dispenses washing liquid. If you have encountered a high background before, then do not hesitate to increase the washing volume, preferably higher than the volume of the coating. Too little washing liquid will make a part of the analysis surface unable to be washed, which will significantly increase the background. The washing volume of all reaction wells must be the same. The manufacturer of the ELISA plate usually lists the coating amount in the manual of the kit. The coating volume commonly used in the industry is 200 μl. If this is the case for your kit, the manufacturer may recommend washing with 300 μl washing solution to clean the walls of the entire reaction well. Generally speaking, the higher the washing volume, the less the amount of antibody or antigen remaining in the incubation step.

The rule of thumb for washing times

The second main parameter that affects the washing effect is the amount of washing cycle. Of course, the more washing times, the lower the background. However, too many washes will reduce the signal intensity, making it difficult to measure. The usual practice is to repeat the washing three times after each antibody or antigen incubation. However, the manufacturer of the ELISA plate will advise on the number of washes. Generally speaking, the number of washes required for the manufacturer’s coated plates is less than that of the user’s coated plates. For user-coated ELISA plates, the number of washes must be optimized.

Another way to control the amount of washing and the number of washings is to add an excess of washing liquid. Generally speaking, each well of a 96-well plate can hold 330 to 460 μl. However, some automated plate washer can be programmed to dispense washing liquid far beyond this amount, such as 1 ml. How does it do it? In fact, it is very simple, that is, turn on the suction function at the same time as the liquid is dispensed. In other words, as the dispenser dispenses more liquid, the aspirator also sucks out the liquid. This technology can increase the washing capacity, but will not spill into other holes.

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Suction

In addition, there are some parameters that will affect the effect of the washing step. These parameters include suction height and suction position, both of which can be adjusted to reduce background (residual amount) and differences.

The remaining liquid contains unbound antigens or antibodies, which increase the background signal. The smaller the residual amount, the less residual liquid, and the less interfering liquid transferred to the next step.

The height of aspiration will obviously affect the residual amount; if the distance between the washing tip and the bottom of the reaction well is slightly larger, the residual amount will increase sharply. Conversely, if the washing tip is pressed against the bottom of the reaction well, it will also reduce the efficiency of liquid absorption and increase the residual volume.

Current plate washing machines generally use two types of shampoos: rigid and floating. The suction head in the floating shampoo can move up and down freely during the washing process, while the rigid shampoo is fixed in place. The washing operation with rigid shampoo is more complicated to optimize, because you need to adjust the suction height very carefully. If your laboratory uses several different boards, it may be time-consuming. When using a floating shampoo, the tip will drop to the bottom of the reaction well. Adjusting the height is not very important, so the shampoo will drop to the bottom.

The suction position also has an important influence on the residual volume; if only one suction point is used per hole (this is common because it is faster), then the suction position needs to be optimized. The best position depends on the characteristics of the shampoo, but it is almost not in the middle of the reaction well, even though this is the most common default position for all shampoos. Generally speaking, the best position is somewhere between the middle of the hole and the wall of the hole.

The shape of the reaction hole also affects the amount of residue. Microplates with a C-shaped bottom (flat-bottomed and rounded corners) generally have lower residual levels than those with flat-bottomed and sharp-cornered microtiters.

If there is no automatic plate washer and manual washing is used, there are several points to note. Use 8-channel or 12-channel micropipettes, and use the backward suction method for washing; washing solutions from different manufacturers should not be mixed with each other. When washing the plate, ensure that the microplate is placed flat and fill the wells with the washing liquid, but try to avoid leakage and overflow, preferably 300 μl per well; the number of plate washes is generally 3 times, and the soaking time of the plate should be guaranteed. The sub-soaking time is generally 30-60 seconds; try to reduce the residual amount of washing liquid. It is recommended to clap the plate after each wash and replace the absorbent paper in time to avoid the debris of the absorbent paper sticking to the reaction well.

ELISA seems very simple, but in the actual operation process, it should not be taken lightly. The washing steps should be checked carefully to obtain accurate results.