• High background

    1;Contamination from other specimens or positive controls:     Repeat ELISA assay, be careful when washing and pipetting

    2;Non-specific binding of antibody:      Use suitable blocking buffers

    3;Incorrect dilutions,eg.,conjugate concentration was too high:     Try different dilutions for optimal results

    4;Longer incubation times and higher incubation temperature than recommended:     Please follow the protocol carefully for  incubation time and temperature of each step

    5;Substrate exposed to light prior to use:     The substrate solution should be kept in dark. Limit exposure to light while running assay
  • The positive control have a weak signal

    1;Reagents did not balance to room temperature:     Ensure reagents and samples are at room temperature before use

    2;Insufficient volum of sample or Incubation time:     Check and follow protocol recommendations

    3;Enzyme inhibitors present in buffer; eg. sodium azide in the washing buffer and assay diluent inhibits peroxidase activity:     Make sure samples and buffer contain no enzyme inhibitor
  • Edge effects

    1;Evaporation:     Use plate sealers between each step.

    2;Uneven temperature:     Place plates in an incubator during incubation periods to avoid temperature fluctuations. Do not stack plates
  • Poor standard curve

    1;Not mixing the standard:     Each step of a dilution standard must be mixing.

    2;Dilutions made too early:     Prepare reagents immediately prior to use and add to wells promptly.

    3;Insufficient standard volum:     Use calibrated pipettes and proper pipetting technique.

  • Poor replicate data

    1;Insufficient washing:      Make sure washing produce is done correctly.

    2;Insufficient mixing of reagents:     nsure all reagents are mixed thoroughly.

    3;Unclean wells:     Wipe bottom of plate to remove any debris or fingerprints prior to reading.

    4;Plate sealers reused:     Use fresh plate sealer for each step.

  • The whole plate generates positive or blue

    1;Insufficient washing:   Check and follow protocol recommendations for washing procedure

    2;Too much streptavidin-HRP:   Check and follow protocol recommendations about dilution and titrate

    3;Substrate Solution mixed too early and turned blue:   Substrate Solution should be mixed and used immediately

  • Weak or no signal

    1;Incubation time too short:   Check and follow protocol recommendations

    2;Incubation temperature too low:   Add stop solution and then determine the optical density using a microplate reader at OD450 nm

    3;Not adding stop buffer
  • Slow color development

    1;Contaminated solutions:  Make fresh solutions

    2;Plates and reagents are at wrong temperature:  Ensure plates are at room temperature and that         the reagents are at room   temperature before use

    3;Detection reagent too old, contaminated or used at the wrong pH:  Use fresh detection reagents        at the correct pH

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