One of the key aspects of optimizing ELISA is washing. The washing step can reduce the background signal caused by unbound antibodies, thereby increasing the signal-to-noise ratio of the analysis. The washing between each step ensures that only specific binding events are retained, and a signal is generated in the final step. Insufficient washing can lead to differences and high background, resulting in undesirable outcomes. This article will introduce some techniques for washing ELISA plates using an automatic washing machine.

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Immunoassay, such as ELISA, typically involves two or more incubation steps separated by washing. ELISA is usually carried out in a 96 well plate, which is coated with binding antigens or antibodies. After the sealing step, the coated plates are first incubated with primary antibodies or antigens. Subsequently, unbound (low affinity) antigen antibody complexes are removed through some washing steps. Next, incubate the plates with secondary antibodies. This enzyme carrying antibody binds to high affinity antigen antibody complexes. Afterwards, there was another round of washing. Finally, add the substrate and detect the signal.

We can use multiple substrates to detect the final antigen antibody complex. There are three types of substrates: natural luminescence, chemiluminescence, and fluorescence. More precisely, the term ELISA refers to the analysis using naturally luminescent substrates. The analysis using chemiluminescent substrates is called Luminescent Immunoassay (LIA), while the analysis using fluorescent secondary antibodies is called Fluorescent Immunoassay (FIA).

1. Washing parameters:

The first main parameter is the amount of washing. The automatic board washer distributes washing solution. If you have encountered a high background before, don't hesitate to increase the washing volume, preferably higher than the package volume. Too little washing solution will prevent some of the analyzed surfaces from being washed, thereby significantly increasing the background. The washing amount of all reaction wells must be the same.

The manufacturer of ELISA plates usually lists the coating amount in the user manual of the kit. The commonly used coating amount in the industry is 200 µ l. If this is also the case with your reagent kit, the manufacturer may recommend using 300 µ l of detergent for washing to clean the entire wall of the reaction well. Generally speaking, the higher the washing amount, the less residual antibodies or antigens remain in the incubation step.

2. Washing frequency:

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, excessive washing can reduce signal strength, making it difficult to measure. The usual practice is to repeat washing three times after each antibody or antigen incubation. However, the manufacturer of ELISA plates will provide recommendations on the number of washings. Generally speaking, the number of washes required for manufacturer packaged tablets is less than for user packaged tablets. For user packaged ELISA plates, the washing frequency must be optimized.

Another method to control the amount and frequency of washing is to add an excess of washing solution. Generally speaking, each hole of a 96 well plate can accommodate 330 to 460 µ l. However, some automated washing machines can be programmed to dispense washing solutions far beyond this amount, such as 1 ml. How is this achieved? It's actually quite simple, just turn on the suction function while separating the liquid. In other words, as the distributor distributes more liquid, the suction device also draws out the liquid. This technology can increase the washing volume, but it will not overflow into other holes.

3. Suction:

Additionally, there are some parameters that can affect the effectiveness of the washing process. These parameters include suction height and suction position, both of which can be adjusted to reduce background (residual amount) and differences.

The residual liquid contains unbound antigens or antibodies, which can increase the background signal. The smaller the residual amount, the less residual liquid there will be, and the less interference liquid will be transferred to the next step.

The suction height will significantly affect the residual amount; If the distance between the washing suction head and the bottom of the reaction hole is slightly larger, it will cause a sharp increase in residual amount. On the contrary, if the washing suction head presses against the bottom of the reaction hole, it will also reduce the liquid absorption efficiency and increase the residual amount.

At present, two types of washing machines are generally used for washing hair: 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 the appropriate position. The washing operation using rigid shampoo is more complex to optimize, as 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 suction head will lower to the bottom of the reaction hole. Adjusting the height is not very important, so washing hair will lower to the bottom.

The position of suction also has a significant impact on the residual amount; If only one suction point is used for each hole (which is common because it is faster), then the suction position needs to be optimized. The optimal position depends on the characteristics of the shampoo, but it is almost not in the middle of the reaction hole, even though this is the most common default position for all shampoos. Generally speaking, the optimal position is somewhere between the middle of the hole and the hole wall.

The shape of the reaction pore can also affect the residual amount. Micro porous plates with C-shaped bottoms (flat bottomed rounded corners) generally have lower residual amounts than those with flat bottomed pointed corners.

If there is no automatic washing machine and manual washing is used, there are also several points to pay attention to. Use an 8-channel or 12 channel micropipette and wash by suction; Washing solutions from different manufacturers should not be mixed with each other. When washing the plate, it is necessary to ensure that the microporous plate is placed flat, and fill each hole with washing solution, but try to avoid leakage and overflow as much as possible, with 300 µ l per hole being appropriate; The number of times the board is washed is generally 3 times, and the soaking time of the board should be ensured. The soaking time for each time is usually 30-60 seconds; Try to minimize the residual amount of washing solution. It is recommended to pat the plate after each wash and replace the absorbent paper in a timely manner to avoid debris from the absorbent paper sticking to the reaction holes.

ELISA may seem simple, but in practical operation, we should not take it lightly. We should still carefully check the washing steps to obtain accurate results.

The above article is excerpted from the internet

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