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Hengyuan Biotechnology | Key points of ELISA experiment - incubation
The incubation in ELISA (enzyme-linked immunosorbent assay) experiments is a crucial step that directly affects the binding efficiency of antigens and antibodies on the solid surface, thereby affecting the accuracy and reliability of the entire experiment. Here are the detailed points about incubation in ELISA experiments. Let's take a look together!
1、 The definition and importance of nurturing
Incubation is the process of antigen antibody binding reaction on the solid surface in ELISA assay, which requires a certain temperature condition for a certain period of time to ensure that the antigen and antibody can fully bind. Incubation is the most critical factor affecting the success or failure of ELISA assays.
2、 Common incubation temperature
1. 43 ℃: To accelerate the reaction, some experiments are conducted at 43 ℃, but higher temperatures should not be used to avoid affecting the specificity of the reaction.
2. 37 ℃: The commonly used insulation temperature in the laboratory, which is also the appropriate temperature for most antigen antibody binding. When establishing ELISA method for reaction kinetics research, the two antigen antibody reactions generally reach their peak in product generation after 1-2 hours at 37 ℃.
3. Room temperature: The standard room temperature refers to 20-25 ℃, but the specific operation can be controlled according to the instructions. When incubating at room temperature, the ELISA plate only needs to be placed flat on the operating table. Attention should be paid to the reaction of room temperature incubation, and the room temperature during operation should be strictly limited within the specified range.
4. 4 ℃ (refrigerator temperature): The antigen antibody reaction is more thorough at 4 ℃. In radioimmunoassay, the reaction is often left overnight in the refrigerator to form the most precipitate. However, due to the long required time, it is generally not used in ELISA.
3、 Incubation time
The reaction incubation time for domestic ELISA kits is usually 30 minutes to 1 hour at 37 ℃.
Imported ELISA kits may require a longer time, such as 1-2 hours at 37 ℃, to achieve more complete binding. Below this time, it may affect the lower limit of the measurement.
4、 Nurturing methods
1. Water bath method: It can effectively solve the problem of uneven heating and avoid edge effects. When using the water bath method, the ELISA plate can be placed in a water bath, and the bottom of the plate should be pressed against the water surface to quickly balance the temperature. To avoid evaporation, the plate should be covered with a lid, or plastic wrap or cling film can be used to cover the plate holes, allowing the reaction plate to float on the water surface.
2. Wet box method: suitable for insulation at low room temperatures, but attention should be paid to avoiding water vapor affecting readings. If a temperature chamber is used, the ELISA plate should be placed in a wet box. The wet box should be made of a material with good heat transfer properties (such as metal), and a wet gauze should be placed at the bottom of the box. Finally, the ELISA plate should be placed on the wet gauze. The wet box should be preheated to the specified temperature in an insulated box, especially when the temperature is low.
3. Microwave radiation method: Use with caution to avoid affecting the experimental results.
5、 Precautions in practical operation
1. Ensure temperature stability: During incubation, it is necessary to ensure that the reaction system is under constant temperature conditions. If using a water bath or incubator for incubation, it is necessary to preheat the incubator to the set temperature and ensure that the ELISA plate is not affected by external temperature fluctuations during the incubation process.
2. Avoid cross contamination: During sample addition and incubation, it is necessary to avoid cross contamination between different samples. When adding samples, a clean pipette tip should be used and each sample should be reacted in an independent well.
3. Pay attention to the heating time: especially when the room temperature is low, it is necessary to extend the Flat noodles's placing time in the temperature box to ensure that the temperature in the hole reaches the set value.
4. Reduce non-specific binding: To minimize the impact of non-specific binding on experimental results, the ELISA plate can be blocked before incubation to block the non-specific binding sites on the plate.
In summary, the incubation step in ELISA experiments requires strict control of temperature, time, and method to ensure sufficient binding of antigens and antibodies, thereby improving the accuracy and reliability of the experiment. Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory and a professional technical research and development team. It is committed to the research and sales of various biological reagents, cells, serum, ELISA kits, and provides free testing services for experiments. We also offer exquisite technical services. If you have any experimental questions, please feel free to consult us and we will provide you with professional one-on-one technical guidance.
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