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- Allow the plate to equilibrate at room temperature for 5 to 10 minutes prior to starting the assay.
- Carry out the assays at approximately 25oC.
- Ensure that wash apparatus is working correctly, recalibrate if necessary.
- Change pipette tips between each standard, sample or reagent. Use separate reservoirs each reagent.
- Wells should appear dry after aspiration.
- Ensure adequate mixing of reagents.
- Adhere to recommended incubation periods and equipment as far as possible.
- Once started, the assay should be completed. Have all standards / samples prepared appropriately before commencement of the assay.
- Prepare substrate(s) within the time recommended in the kit insert.
- Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the kit insert.
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- Possibility of inadequate volume of substrate added to the wells.
- Incomplete incubation of plate after adding the substrate solution.
- The conjugate concentration may be different than that recommended.
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- Errors in incubation times or temperature changes may lead to absence of color development.
- It is necessary to read the plate at the correct calorimetric wavelength within 15 minutes after adding the stop solution.
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- Incomplete washing of wells with less than the recommended amount of buffer can result in high background. Please follow the washing instruction given in the insert.
- Incubation of plate for very long time or higher than recommended temperature can result in high background.
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- It is possible due to interrupted assay set up or reagents not at appropriate room temperature.
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- Unequal washing of wells during washing steps.
- Inadequate mixing of reagents.
- Wells should be aspirated carefully. Tap the plates wells down on paper towels after the last wash to ensure residual moisture is removed.
- Do not prepare the substrate too early. Follow the time recommended in the kit insert.
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- While loading the antibodies in the plate, avoid creating bubbles in the wells.
- If the % CV values for the standard curve OD values are consistently above 15% pay particular attention to pipetting technique. If standard curve ODs are acceptable but sample precision is not, follow the recommendations to improve precision.
- Washing the plate between incubation steps should be uniform and each well should be filled completely with wash buffer.
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- Fluctuations in temperature and humidity around the work surface can lead to edge effects.
- Edge effects could also arise due to inadequate fixing of plate covers, leading to evaporation.
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