Here is a trouble-shooting check-list:
Did you correctly follow the instructions for use (incubation times, dilutions, number of washes, preparation of 1X solutions)?
Has the washing system been properly maintained and regularly decontaminated?
What water was used to prepare the wash solution? We recommend using water which is distilled or purified by reverse osmosis or a system such as MilliQ or Elga, which reduces the amount of bacteria in the water.
Were any kit reagents inter-mixed or contaminated?
Was the dishware clean?
Was the substrate solution transparent before use?
Has the kit expired?
Were all reagents and components brought up to room temperature 18-25ºC before running the assay?
What is the room temperature in the lab? It should be between 18-25ºC. As ELISA tests are sensitive to extreme temperature, avoid running the assay close to heating sources.
Have you ensured that the pipettes are dispensing the correct volumes, and that the pipette tips are always tightly on before use?
Did you read the plates at the correct wavelength?
Were previously opened plates correctly stored? Please refer to Plate storage.pdf
Were the storage conditions respected for all components, including any 1X solutions made from concentrated solutions? Please refer to Storage conditions.pdf.