Before the incubation process, the sample loading time can be too long, which may lead to delays in adding the enzyme reagent. When using a dropper to add the reagent, the angle and pressure of the drop can vary, resulting in inconsistent volumes being added. This inconsistency can affect the accuracy of the test results.
Control Measures:
1. If there are many samples to process, it is advisable to work in batches to minimize errors caused by extended processing times.
2. After adding the enzyme reagent, use absorbent paper to gently remove any excess reagent that may remain on the well's edges.
3. For quantitative tests, use a calibrated pipette to ensure accurate reagent delivery. Regularly check and calibrate the pipette for accuracy, and always use separate tips for different samples to avoid cross-contamination.
Incubation is one of the most critical and challenging steps in ELISA assays. Common incubation temperatures include 37°C, room temperature, 43°C, and 4–8°C. Most domestic ELISA kits recommend an incubation at 37°C for 30 minutes to 1 hour, while imported kits often require 1 to 2 hours at the same temperature to achieve a more complete reaction.
The incubation time and temperature should generally follow the kit’s instructions. Once the sample and/or reagent are added, the microplate is placed in a water bath or incubator. It takes some time for the temperature in the wells to rise from room temperature to 37°C. If timing starts immediately after placing the plate in the incubator, the actual incubation time may be shorter than intended, potentially leading to underdevelopment, especially in weak positive samples.
Control Measures:
1. If possible, opt for a slightly lower temperature with a longer incubation time to ensure a more uniform and complete reaction.
2. Place a small thermometer inside the plate during incubation to monitor the actual temperature.
3. Be aware of the phenomenon where the outer wells develop more intensely than the central ones due to thermal gradients. To reduce this effect, consider using a water bath instead of an incubator for more even heating.
By carefully managing these factors, you can improve the consistency and reliability of your ELISA results.
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