Enzyme-linked solvent assay (ELISA) is a very useful and popular method to analyze biological analytes. However, we sometimes have problems with not paying attention each step.
Several steps below must be checked when the OD values are very low.
In sandwich assays, the following may need to be checked.
- Capture antibody lost activity.
- Capture antibody was prepared at the wrong concentration.
- Capture antibody was not coated during incubation.
- Wrong buffers were used for diluting samples/standards or detection antibody
- Detection antibody lost activity.
- Detection antibody was prepared at the wrong concentration.
- Chromogen received oxidation before assay.
- Plates were read with an inappropriate filter