Experts from Creative Biomart Share the Experience on Using Elisa Kits to Improve the Precision of an Assay
There are some issues that researchers might meet in the process of using ELISA Kits. In order to help improve the precision of an assay, Creative Biomart (http://www.creativebiomart.net/) experts concluded the following tips for reference.
New York, NY, August 14, 2013 --(PR.com)-- 1. Positive results in negative control, might be caused by:
Contamination of reagents/samples—use fresh reagents and pipette carefully.
Insufficient washing of plates—ensure wells area washed adequately by filling the wells with wash buffer. Ensure all residual antibody solutions are removed before washing.
Too much antibody used leading to non-specific binding—check the recommended amount of antibody suggested. Try using fewer antibodies.
2. High background across entire plate, might be caused by:
Conjugate too strong or left on too long—check dilution of conjugate, use it at the recommended dilution. Stop the reaction using stop buffer as soon as the plate has developed enough for absorbance readings.
Substrate solution or stop solution is not fresh—use fresh substrate solution. Stop solution should be clear.
Reaction not stopped.
Plate left too long before reading on the plate reader.
Contaminants from laboratory glassware—Sterilize glassware beforehand if Substrate incubation carried out in the light.
Non-specific binding of antibody—ensure a block step is included and a suitable blocking buffer is being used.
3. Low absorbance values, might be caused by:
Target protein not expressed in sample used/ or Low level of target protein—check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay.
Insufficient antibody—check the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.
Substrate solutions not fresh or combined incorrectly—prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed at the correct concentration.
Reagents not fresh or not at the correct pH—ensure reagents have been prepared correctly and are in date.
Incubation time not long enough—ensure you are incubating the antibody for the recommended amount of time, if an incubation time is suggested.
Incubation temperature too low—Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working.
Stop solution not added.
4. High absorbance values for samples and/or positive control - absorbance does not go down as the sample is diluted down the plate), might be caused by:
The concentration of samples or positive control is too high and out of range for the sensitivity of the assay. —Re-assess the assay you are using OR reduce the concentration of samples and control by dilution before adding to the plate. Take the dilution into account when calculating the resulting concentrations.
5. Inconsistent absorbances across the plate, might be caused by:
Plates stacked during incubations.
Pipetting inconsistent—ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid.
Antibody dilutions/Reagents not well mixed—ensure a consistent concentration across all wells, ensure all reagents and samples are mixed before pipetting onto the plate.
Wells allowed to dry out—ensure lids are left on the plates at all times when incubating. Place a humidifying water tray (bottled clean/sterile water) in the bottom of the incubator.
Inadequate washing.
Bottom of the plate is dirty affecting absorbance readings.
6. Color developing slowly, might be caused by:
Plates are not at the correct temperature—Ensure plates are at room temperature and that the reagents are at room temperature before use.
Conjugate too weak—prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed, at the correct concentration.
Contamination of solutions—Presence of contaminants, such as sodium azide and peroxidise can affect the substrate reaction. Avoid using reagents containing these preservatives.
Creative Biomart is a world leading supplier that offers a wide variety of complete ELISA kits that have been exhaustively tested for superior quality and reproducibility. They are available in a range of formats including colorimetric, fluorescence, and chemiluminescence-based kits for various types of Analyte. For additional information, please contact info@creative-biomart.com or visit http://www.creativebiomart.net/ELISA_kits.htm.
Contamination of reagents/samples—use fresh reagents and pipette carefully.
Insufficient washing of plates—ensure wells area washed adequately by filling the wells with wash buffer. Ensure all residual antibody solutions are removed before washing.
Too much antibody used leading to non-specific binding—check the recommended amount of antibody suggested. Try using fewer antibodies.
2. High background across entire plate, might be caused by:
Conjugate too strong or left on too long—check dilution of conjugate, use it at the recommended dilution. Stop the reaction using stop buffer as soon as the plate has developed enough for absorbance readings.
Substrate solution or stop solution is not fresh—use fresh substrate solution. Stop solution should be clear.
Reaction not stopped.
Plate left too long before reading on the plate reader.
Contaminants from laboratory glassware—Sterilize glassware beforehand if Substrate incubation carried out in the light.
Non-specific binding of antibody—ensure a block step is included and a suitable blocking buffer is being used.
3. Low absorbance values, might be caused by:
Target protein not expressed in sample used/ or Low level of target protein—check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay.
Insufficient antibody—check the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.
Substrate solutions not fresh or combined incorrectly—prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed at the correct concentration.
Reagents not fresh or not at the correct pH—ensure reagents have been prepared correctly and are in date.
Incubation time not long enough—ensure you are incubating the antibody for the recommended amount of time, if an incubation time is suggested.
Incubation temperature too low—Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working.
Stop solution not added.
4. High absorbance values for samples and/or positive control - absorbance does not go down as the sample is diluted down the plate), might be caused by:
The concentration of samples or positive control is too high and out of range for the sensitivity of the assay. —Re-assess the assay you are using OR reduce the concentration of samples and control by dilution before adding to the plate. Take the dilution into account when calculating the resulting concentrations.
5. Inconsistent absorbances across the plate, might be caused by:
Plates stacked during incubations.
Pipetting inconsistent—ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid.
Antibody dilutions/Reagents not well mixed—ensure a consistent concentration across all wells, ensure all reagents and samples are mixed before pipetting onto the plate.
Wells allowed to dry out—ensure lids are left on the plates at all times when incubating. Place a humidifying water tray (bottled clean/sterile water) in the bottom of the incubator.
Inadequate washing.
Bottom of the plate is dirty affecting absorbance readings.
6. Color developing slowly, might be caused by:
Plates are not at the correct temperature—Ensure plates are at room temperature and that the reagents are at room temperature before use.
Conjugate too weak—prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed, at the correct concentration.
Contamination of solutions—Presence of contaminants, such as sodium azide and peroxidise can affect the substrate reaction. Avoid using reagents containing these preservatives.
Creative Biomart is a world leading supplier that offers a wide variety of complete ELISA kits that have been exhaustively tested for superior quality and reproducibility. They are available in a range of formats including colorimetric, fluorescence, and chemiluminescence-based kits for various types of Analyte. For additional information, please contact info@creative-biomart.com or visit http://www.creativebiomart.net/ELISA_kits.htm.
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Creative Biomart
Dynah Luo
(1)-516-669-8109
http://www.creativebiomart.net/
Dynah Luo
(1)-516-669-8109
http://www.creativebiomart.net/
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