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How To Wash ELISA Microplate?

Posted on October 29, 2022 by Zeng YingELISA

Catalogue: Equipment For Elisa Kit Manufacturing

ELISA Microplate - directly affects ELISA results

One of the critical points in optimizing ELISA is the washing of the ELISA microplate. the ELISA plate-washing step reduces the background signal caused by unbound antibodies, thus increasing the signal-to-noise ratio of the analysis. Washing between degrees ensures that only specific binding events are retained, generating a signal in the final step. Inadequate washing can lead to discrepancies and high background, resulting in suboptimal results.

When doing ELISA experiments, there are two ways to wash ELISA microplates: manual method wash ELISA plates and plate washers to wash ELISA plates. There is no essential difference between the two, as long as the operation is in line with the requirements, both can get correct and reliable results.

Manual washing of ELISA microplate

ELISA-result
ELISA-result
ELISA plate sealer, PCR plate sealer

Manual washing ELISA plate human factor is relatively large, the experimenter must be experienced, the number of washes to be sufficient, the impact is not too large, adding the amount of washing solution to just full reaction wells as a limit to prevent the pore mouth with free enzyme failed to wash, while preventing the liquid cross between the pore and the hole. It is also very important to withhold dry after washing, otherwise, it is easy to cause false positives. After each wash, gently pat the plate dry on absorbent paper, and pat the plate vertically to avoid cross-infection. The force should not be too violent to prevent the antigen-antibody complex from detaching; the absorbent paper should be used once, and absorbent paper that is not easy to shed debris should be used as appropriate. The enzyme conjugate is not resistant to drying, especially at higher temperatures, it is more likely to be inactivated, so the dried reaction plate should be patted to shorten the exposure time in the air as much as possible, the longer the time, the lower the absorbance value.

Manual cleaning of enzyme-linked immunosorbent assay microplates is performed by both immersion and flow-through methods

Immersion

1. Aspirate or shake dry the reaction solution in the ELISA microplate.

2. Wash the plate once with washing solution (after filling the wells with washing solution, then shake off).

3. Soak, i.e. fill the wells with washing solution, place for 1~2 minutes, shake intermittently, soaking time should not be shortened arbitrarily.

4. Suction dry the liquid in the hole. Suction dry should be thorough, available pump or vacuum pump suction, can also be shaken off the liquid in a clean towel or absorbent paper pat dry.

5. Repeat operation steps 3 and 4, washing 3 to 4 times (or as specified in the instructions). In the indirect method if the background is higher, you can increase the number of washes or extend the soaking time.
Micro titration plate is mostly used by immersion washing method. The washing solution is mostly a neutral buffer containing non-ionic detergent. The combination of polystyrene carrier and protein is hydrophobic, and the non-ionic detergent contains both hydrophobic and hydrophilic groups, whose hydrophobic groups are combined with hydrophobic groups of protein by hydrophobic bonds, thus weakening the combination of protein and solid-phase carrier, and with the combination of hydrophilic groups and water molecules, the protein returns to the state of aqueous solution, and is thus separated from the solid-phase carrier.
The non-ionic detergent in the washing solution is generally Tween 20, the concentration of which can be between 0.05% and 0.2%, higher than 0.2%, can make the antigen or antibody encapsulated in the solid phase desorption and reduce the sensitivity of the test.

Flowing water flushing type

The flowing water rinsing method is initially used for washing small bead carriers, and the washing solution is only distilled water or even tap water. When washing, a special device is attached, so that the small beads are continuously rolled and showered under the impact of running water, and after continuous rinsing for 2 minutes, the liquid is drained, and then soaked in distilled water for 2 minutes and drained. Soaking type is like a tub bath, water rinsing type is like a shower, its washing effect is more thorough, and also simple, fast.

Experiments have shown that the rinsing type is also applicable to the washing of ELISA micro titration plates. When washing, try to increase the water flow or increase the water pressure, let the water impact the surface of the plate hole, the washing effect is better.

Plate washer for ELISA microplate

ELISA-plate
ELISA-plate

The plate washer washes ELISA plates with few human factors, fast and constant conditions, but the following three key points should be noted when using the plate washer to wash plates.

Parameters for washing ELISA plates

The main parameter for washing ELISA microplates is the wash volume. The automatic plate washer dispenses the wash solution. If you have experienced high background before, then do not hesitate to set the wash volume to high, preferably higher than the envelope volume. Too little wash solution will leave a portion of the analyzed surface unwashed, thus significantly increasing the background. The wash volume must be the same for all reaction wells.

Number of washing cycles in ELISA plate

The main parameter that affects the effectiveness of washing ELISA microplates is the amount of washing cycles. Of course, the higher the number of washes, the lower the background. However, too many washes can reduce the signal intensity and make it difficult to assay. It is common practice to repeat the wash three times after each antibody or antigen incubation. However, in general, wrapped plates require fewer washes than user-wrapped plates. For user-coated ELISA microplates, the frequency of washes must be optimized.

Another way to control the amount and number of washes is to add an excess of wash solution. Typically, a 96-well plate holds 330 to 460 µl per well. however, some automated plate washers can be programmed to dispense much more than this amount of wash solution, for example 1 ml. how does it do this? It is actually quite simple: the aspiration function is turned on at the same time as dispensing. In other words, as the dispenser dispenses more liquid, the aspirator also sucks the liquid out. This technique increases the amount of wash, but without spilling into other wells.

Aspiration of ELISA microplates

ELISA-test-kit
ELISA-test-kit

There are also a number of parameters that affect the effectiveness of the washing step of an ELISA plate. These parameters include aspiration height and aspiration position, both of which can be adjusted to reduce background (residual volume) and variation.

Residual liquids contain unbound antigens or antibodies, which increase the background signal. The smaller the residual volume, the less residual liquid there is and the less interfering liquid is transferred to the next step.

The height of the aspirate can significantly affect the residual volume; if the distance between the wash tip and the bottom of the reaction well is slightly larger, then the residual volume will increase dramatically. Conversely, if the washing tip is pressed against the bottom of the reaction well, then it will also make the aspiration less efficient and the residual amount will increase.

Current machines for washing ELISA plates generally use two types of wash heads: rigid and floating. The suction head in a floating wash head can move freely up and down during the washing process, while a rigid wash head is fixed in the proper position. Washing operations with rigid wash heads are more complicated to optimize because you need to adjust the suction height very carefully. If your lab uses several different plates, it can be very time consuming. When using a floating wash head, the pipette tip will drop to the bottom of the reaction well. Adjusting the height would not be very important, so the wash head would drop to the bottom.

The position of the suction also has an important effect on the residual volume; if only one suction point is used per well (which is common because it is faster), then the position of the suction needs to be optimized. The optimal location depends on the trait of the wash head, but it is hardly in the middle of the reaction well, even though this is the most common default location for all washes. In general, the optimal location is somewhere between the middle of the hole and the wall of the hole.

The shape of the reaction wells also affects the residual amount. microplates with C-shaped bottoms (flat bottoms with rounded corners) will generally have lower residual amounts than those with flat bottoms with sharp corners.

Precautions for washing ELISA plate

a. The amount of liquid injection and the amount of residual liquid should be corrected daily, and the amount of residual liquid after washing should not be greater than 2 μl.

b. Replace the broken aspiration needle in time to avoid damaging the substrate coating.

c. Pay attention to adjust the drop height of the aspiration head for different manufacturers of enzyme plates to avoid the rinse needle from jamming the plate.

d. Pay attention to adjust the amount of washing solution, too much washing solution will overflow and too little will not achieve the washing effect.

e. Use distilled water to flush the pipeline before shutting down the machine to avoid the crystalline matter of the washing solution from blocking the pipeline.

f. It is strictly forbidden to mix the washing solutions of different kits.

Conclusion

In order to make the ELISA plate washing effect better, the laboratory can use a combination of machine and manual washing method, after the machine washing and then manual washing plate 1 to 2 times, can achieve the ideal washing effect. The results are guaranteed!

Use distilled water to flush the pipeline before shutting down the machine to avoid the crystalline matter of the washing solution from blocking the pipeline.

It is strictly forbidden to mix the washing solutions of different kits.

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