ELISA Troubleshooting

Weak standard curve signals


If the standard curve OD signals are low (highest standard < 1.0), please consider the following tips and best practices:

  • Standard improper reconstitution. It is critical to briefly centrifuge the vials of Standard before opening. After reconstituting the standard with Assay Diluent, the liquid may be gently mixed by either pipetting up and down. The vial should then be centrifuged briefly to collect the liquid at the bottom of the tube. Vortexing is generally not recommended.
  • Inaccurate dilution of the Detection Antibody or HRP-Streptavidin. Double check the preparation of these reagents, as even a slight variation can produce a noticeable difference in signal.
  • Short incubation time: Using shorter incubation times than what is recommended in the manual can cause lower signals. Conversely, incubating the standards overnight at 4˚C can boost the overall signals on the microplate.
  • Bioaim recommends preparing a fresh stock of standard and retest using the tips above.

Low overall signal


  • Detection Antibody – Be sure to centrifuge the vial before open the lid. Once reconstituted, this reagent must be used within a day or two.
  • HRP-Streptavidin – Be sure to centrifuge the vial before open the lid. Pipet up and down to mix thoroughly, don’t vortex it rigorously. After dilution, the HRP-streptavidin solution must be used the same day. Discard the unused portion.
  • TMB One-Step Substrate - This component is stable for 12 months at 4˚C, but must be protected from light exposure.
  • Pre-coated Microplate – After opening the moisture barrier bag, any unused microplate strips must be stored at 4°C and used within 1 month. Return unused strips to the bag containing the desiccant pack, and reseal the bag along the entire edge.
  • Adhere to the kit storage guidelines (4˚C for 6 months or -20˚C for 1 year). Avoid multiple freeze-thaw cycles.

High Background


Common causes of high background include:

  • Insufficient wash: if the plate is insufficiently washed or if the Wash Buffer is contaminated. Wash Buffer must be removed completely from the wells after each wash. An automated plate washer or multi-channel pipettor is recommended for best results.
  • Too much HRP-Streptavidin: Follow the manual to get the correct HRP-Streptavidin dilution.
  • Too much Detection Antibody: Ensure the correct dilution of Biotinylated Antibody is used.

High CV in replicate wells


Common causes of high well-to-well variability include:

  • Incomplete washing: ensure complete removal of wash buffer after each wash.
  • Pipet performance: be sure pipets are calibrated and function properly.
  • Bubbles in wells: Try to avoid any bubbles during sample/ reagent pipetting.

Matrix effects

This can occur when proteins or other components within the sample affect the immunoreactivity of the target molecule. These matrix components can also affect the ability of the antibody to recognize its target within the sample. Auto-antibodies, binding proteins, hemolysis, or certain disease states can contribute to this phenomenon. If matrix effects are suspected, centrifuge the sample and dilute further. When testing serum or plasma, a minimum 2-fold dilution is always recommended to avoid matrix effects.