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 step5;Substrate exposed to light prior to use: The substrate solution should be kept in dark. Limit exposure to light while running assay
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 recommendations3;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
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
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.
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.
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
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 nm3;Not adding stop buffer
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