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.