ELISA is the abbreviation for Enzyme-Linked Immunosorbnent Assay. It is an immunoenzyme technology developed after immunofluorescence and radioimmunoassay. This technology has developed very rapidly since its introduction in the early 1970s, and it has been widely used in many fields of biology and medical sciences.
Incubation is one of the most critical factors affecting the success or failure of the ELISA.
The time required for incubation is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperature is 37 ℃ and room temperature, followed by 43 ℃ and 2 ~ 8 ℃. The incubation step is the most problematic step in the ELISA assay. Generally, the reaction incubation time of the domestic ELISA commercial kits is currently 37 ° C for 30 minutes to 1 hour, then it is usually 37 ° C for 1 to 2 hours to have a more complete binding, and less than 1 hour may affect the lower limit of the assay.
Operation notes
1. To ensure that there is sufficient reaction time at the set temperature. Generally speaking, after adding the sample and (or) reaction reagent, when the microplate is taken from the room temperature to the water bath or incubator, the temperature in the well is from A room temperature rise of 37 ° C requires a certain amount of time, especially under low room temperature and non-water bath conditions. This period of heating may be relatively long, and in clinical laboratories, there are few days when the indoor temperature is low. At this time, after the microplate was transferred to the incubator, the 37 ℃ incubation time was not enough, so that the weak positive sample was negative.
2. Selection of incubation temperature In the instructions of some ELISA kits, it is pointed out that there are two kinds of incubation temperatures, for example, one is 37 ° C for 1h, and the other is 43 ° C for 45min. Therefore, we recommend using lower incubation temperatures and longer reaction times in clinical ELISA assays.
3. In the routine ELISA measurement in the laboratory, the plate is placed at room temperature (usually around 25 ° C) in a 37 ° C incubator. When the plate hole is heated, there may be a thermodynamic gradient between the peripheral hole and the central hole. Therefore, a water bath is used or when the reaction solution is added to the wells of the plate, both the plate and the solution are added.
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