TROUBLESHOOTING TIPS - ELISA
Contents
1. 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 Inconsistent absorbances across the plate Plates stacked during incubations Stacking of plates does not allow even distribution of temperature across the wells of the plates Avoid stacking 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 This will greatly affect consistency of results between duplicates Antibody dilutions Reagents not well mixed To 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 This will lead to some wells not being washed as well as others leaving different amounts of unbound antibody behind which will give inconsistent results Bottom of the plate is dirty affecting absorbance readings Clean the bottom2. abcam TROUBLESHOOTING TIPS ELISA Positive results in negative control Contamination of reagents samples May be contamination of reagents or samples or cross contamination from splashing between wells Use fresh reagents and pipette carefully Sandwich ELISA Detection antibody is detecting coating antibody Check the correct coating antibody and detection antibodies are being used and that they will not detect each other 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 To much antibody used leading to non specific binding Check the recommended amount of antibody suggested Try using less antibody High background across entire plate Conjugate to 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 if it has gone yellow this is a sign of contamination and it should be replaced Reaction not stopped Colour will keep developing if the substrate reaction is not stopped Plate left too long before reading on the plate reader Colour will keep developing though at a slower rate if stop solution has been added Contami
3. ck the recommended amount of antibody is being used The concentration of antibody may require increasing for optimisation 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 The incubation time may require increasing for optimisation of results Incubation temperature too low Antibodies will have optimum binding activity at the correct temperature Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working Incubation temperature may require some optimisation www abcam com technical abcam E Ensure all reagents are at room temperature before proceeding Stop solution not added Addition of stop solution increases the intensity of colour reaction and stabilizes the final colour reaction High absorbance values for samples and or positive control absorbance does not go down as the sample is diluted down the plate The concentration of samples or
4. nants from laboratory glassware Ensure reagents are fresh and prepared in clean glassware Sterilise glassware beforehand if Substrate incubation carried out in the light Substrate incubation should be carried out in the dark www abcam com technical abcam E Incubation temperature too high Antibodies will have optimum binding activity at the correct temperature Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working Incubation temperature may require some optimisation Non specific binding of antibody Ensure a block step is included and a suitable blocking buffer is being used We recommend using 5 to 10 serum from the same species of the secondary antibody or bovine serum Ensure wells are pre processed to prevent non specific attachment Use an affinity purified antibody preferably pre absorbed Also check suggestions listed under Positive results in negative control Low absorbance values Target protein not expressed in sample used Low level of target protein expression in sample used 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 Ensure you are using a positive control within the detection range of the assay Insufficient antibody Che
5. of the plate carefully before re reading the plate Colour developing slowly Plates are not at the correct temperature www abcam com technical abcam 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 www abcam com technical
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