By Ryan Hamnett, PhD
ELISAs use antibodies to detect and quantify the amount of a target antigen in a liquid sample. Below is our troubleshooting guide to help solve any issues that might be encountered with an ELISA experiment.
| Potential issue: | Possible solution: |
|---|---|
| Blocking protein in coating solution | Use coating solution containing only buffer components and the coating antigen or antibody |
| Capture antibody does not bind to plate | Use ELISA plate, not a tissue culture plate Try longer coating time Increase concentration of coating antibody Consider using a more active form of coating, such as Protein A/G or metal-coated plates |
| Recognition of epitope is blocked by antigen adsorption to plate | Consider a more active form of antigen coating If assaying a small peptide, try conjugating peptide to a large carrier protein before coating |
| Protein of interest is not present, or is present below detection limit | Run positive control to ensure assay is working correctly Decrease dilution factor, or concentrate samples Check literature to determine if protein of interest is expected in sample type |
| Incompatible sample type | Include positive control to ensure ELISA kits works with your sample type |
| Incubation time too short | Increase incubation time or incubate overnight at 4°C |
| Incubation temperature too low | Ensure all reagents including plate are at room temperature before starting assay Carry out incubation steps according to manufacturer’s instructions |
| Too much washing in between steps | Though washing is essential, washing too aggressively (e.g. with too much pressure) can remove detection reagents. Wash with gentle pressure |
| Wells scratched with pipette tips | Carefully dispense and remove solutions |
| Wells have dried out | Cover the plate for each step using sealing film |
| Primary and secondary antibodies not compatible | Use a secondary antibody that has been raised against the host species for the primary antibody Ensure secondary antibody can recognize the isotype of the primary antibody |
| Insufficient antibody | Optimize antibody concentration |
| Enzyme inhibitors present in buffers | Avoid using buffers containing sodium azide with HRP-based detection Avoid using buffers containing phosphate with AP-based detection |
| Insufficient substrate | Optimize substrate concentration |
| Plate read at incorrect detection wavelength | Use the recommended wavelength and filter settings for the detection system being used |
| Detection system not sufficiently sensitive | Consider using a more sensitive detection system such as chemiluminescence Consider an amplification, such as the biotin-avidin system |
| Sample improperly stored | Store samples at -80°C to avoid protein degradation, or assay immediately after collection and processing |
| Antibodies improperly stored | Store antibodies at 4°C only for short-term usage, otherwise aliquot and store at -20°C Use a fresh aliquot of frozen antibody Avoid freeze-thaw cycles |
| Other reagents have lost activity due to improper handling or storage | Follow manufacturer’s instructions for storage – many components need to be stored at 4°C Generally avoid freeze-thawing Handle fluorophores, fluorophore-conjugated antibodies, and enzyme substrates in the dark Run a positive control to ensure all reagents are working appropriately |
| Buffers contaminated with bacteria | Make up fresh buffer using sterile components |
| Potential issue: | Possible solution: |
|---|---|
| Sample is too concentrated | Determine optimal dilution by titration |
| Substrate changed color before use in assay | Make substrate solution fresh, immediately before use |
| Plate read at incorrect wavelength | Use the recommended wavelength and filter settings for the detection system being used |
| Excess time before plate reading | Read plate shortly after adding substrate Use a stop solution |
| Potential issue: | Possible solution: |
|---|---|
| Incubation time too long | Reduce incubation time Try incubating overnight at 4°C |
| Buffers are contaminated with metals or enzyme | Make up fresh buffers Change pipette tips when pipetting different solutions |
| Insufficient blocking | Increase the blocking incubation time Try an alternative blocking agent, such as 5-10% normal serum from the same species as the enzyme-conjugated antibody |
| Insufficient washing | Use the full volume of wash buffer for each well to ensure complete washing, as previous reagents and sample stick to sides of well Increase number of wash steps At the end of each wash step, flick plate over a sink and then pat on a paper towel to remove as much wash buffer as possible |
| Uneven evaporation of solution from wells | Perform all incubations with a lid or seal on plate |
| Excess antibody | Find optimal antibody concentration by titration |
| Non-specific binding by enzyme-conjugated antibody | Run S0, NSB and secondary antibody controls to confirm source of background Use a primary antibody raised in a different species to the sample Dilute sample to reduce binding to interfering factors and anti-animal antibodies Include Tween-20 in wash buffers to reduce non-specific interactions Optimize incubation temperature |
| Contamination of reagents | Change pipette tips between different solutions Use fresh reagents |
| Excessive substrate used | Follow manufacturer’s instructions for substrate Dilute substrate |
| Precipitate in wells on addition of substrate | Decrease concentration of substrate Increase washing after enzyme-conjugate is added |
| Color development carried out in light | Carry out substrate incubation in dark |
| Wells are the wrong color after stop solution addition | Ensure stop solution is completely mixed with the substrate solution in the well |
| Overdeveloped color | Use stopping solution Read plate shortly after color development |
| Excessive amplification (if using) | Reduce the amount of amplification, such as by conjugating less biotin to antibody |
| Contamination in blank control wells | Change pipette tips between blanks and samples Seal plate or put a lid to reduce spillage between wells |
| Potential issue: | Possible solution: |
|---|---|
| Standards were not diluted correctly | Confirm calculations |
| Standard stock solution not fully reconstituted | Spin down vial before opening; inspect for undissolved material after reconstitution |
| Standard degraded | Ensure proper handling and storage of standard stock |
| Pipetting error | Calibrate pipettes Use proper pipetting technique |
| Standard curve model does not fit data | Try an alternative model, such as semi-log, 4PL, and 5PL plots |
| Flat portions of standard curve | Some standard wells are likely saturated – reduce image acquisition time, or reduce concentrations of reagents in earlier steps |
| Potential issue: | Possible solution: |
|---|---|
| Bubbles in wells | Use proper pipetting technique Remove bubbles with pipette |
| Wells not washed equally | Wash all wells of the plate as recommended Use automatic plate washers |
| Inconsistent pipetting | Use calibrated pipettes and proper pipetting technique Ensure all reagents are completely mixed by pipetting up and down or gently inverting |
| Inconsistent sample preparation and storage | Store all samples under the same conditions, ideally aliquoted at -80°C |
| Particulates in samples | Centrifuge samples to remove any particulate matter |
| Edge effects – outer wells of plate showing different signal to inner wells, usually due to changes in environment | Check if buffer has evaporated from outer wells – if so, ensure plate cover is sealed to prevent evaporation Ensure plate is fully equilibrated to room temperature before beginning – inner wells take longer to reach correct temperature Do not stack plates, which may cause uneven temperature distribution across the plates |