Though without that complicated protocols as IHC, immunoprecipitation etc, ELISA assay might generate many problems as what could occur in most of the immuno assays. This part provides troubleshooting of ELISA with means of cause-solution table with problems listed beforehand. Please note that the conditions described here may not pertain to every ELISA kit because performance requirements vary for individual assays. Be sure to check your package insert for specifications.
(1) If ELISA signal is extraordinarily high, and standard curves have saturated ODs, its possible causes and recommended troubleshooing are:
| Possible causes | Recommended troubleshooting |
|---|---|
| Standard reconstituted with less volume than required. | Reconstitute lyophilized standard with correct volume of solution recommended in the protocol. |
| Plate incubation was too long. | Decrease incubation time. |
| Detection antibody incubation time is too long. | Decrease detection antibody incubation time. |
| Substrate solution incubation time is too long. | Decrease substrate solution incubation time. |
(2) If sample readings are out of range, possible causes and troubleshooting are as follows.
| Possible causes | Recommended troubleshooting |
|---|---|
| Samples contain no or below detectable levels of analyte. | If samples are below detectable levels, it may be possible to use higher sample volume. Check with technical support for appropriate protocol modifications. |
| Samples contain analyte concentrations greater than highest standard point.. | Samples may require dilution and reanalysis. |
As below we lists the most common problems in ELISA assay, click for more details.