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Do you know the correct method of ELISA kit washing?

Views : 1867
Update time : 2023-01-31 16:51:48
ELISA relies on washing for the purpose of separating free and bound enzyme markers. It is done by washing to remove residual substances in the plate wells that do not bind to the solid phase antigen or antibody, as well as interfering substances that are not specifically adsorbed to the solid phase carrier during the reaction. Today, JINMA summarizes the two main methods of ELISA kit washing for you.
Washing methods
Immersion type

1. aspirate or shake dry the reaction solution in the wells.

2. overwash with washing solution (after filling the plate wells with washing solution, that is, shake off).

3. soak, i.e. fill the plate 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 c and d, washing 3-4 times (or as specified in the instructions). In the indirect method such as the background is higher, you can increase the number of washing or extend the immersion time.

Flow rinsing type

Initially used for the washing of small bead carriers, the washing solution is only distilled water or even tap water. When washing, a special device is attached, so that the small beads under the impact of the water constantly rolling shower, continuous rinsing 2 minutes, then absorb the liquid, and then soak in distilled water for 2 minutes, absorb dry. The soaking type is like a tub bath, and the rinsing type is like a shower, which is more thorough and easy and fast. Experiments have shown that the rinsing type is also applicable to the washing of micro titration plates. When washing, we try to increase the water flow or water pressure, so that the water can impact the surface of the plate holes, the washing effect is better.

In addition to the washing method, there are main washing parameters, washing times, etc., which are also crucial in the experiments, and our technology also gives you a summary of the optimization of washing experiments.

ELISA kit washing parameters
First, the amount of washing

The automatic plate washer will distribute the washing solution. If you have previously encountered high background, then do not hesitate to adjust the wash volume to high, preferably higher than the volume of the package. 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, and the manufacturer of the ELISA plate will usually list the overcoat volume in the kit's instruction manual. The usual pack volume used in the industry is 200 μl. If this is also the case for your kit, then the manufacturer may recommend washing with 300 μl of washing solution in order to clean the entire wall of the reaction wells. In general, the higher the wash volume, the lower the amount of antibody or antigen left in the incubation step.

Second, the amount of washing cycles

[ELISA kit washing] the more times, the lower the background. However, too many washes can reduce the signal intensity and make it difficult to measure. It is common practice to repeat the wash three times after each antibody or antigen incubation. However, the manufacturer of the ELISA plate will make recommendations on the number of washes. In general, manufacturer-coated plates require fewer washes than user-coated plates. For user-coated ELISA plates, the number of washes must be optimized.

Another way to control the amount and number of washes is to add an excess of wash solution. Generally, 96-well plates can hold 330 to 460 ul 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 does not spill into other wells.
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