ELISA Troubleshooting Saturated Signal

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ELISA Troubleshooting: Saturated Signal

If your ELISA signal is too high, the results of the experiment can become unusable. Saturated signal can cause wells to appear uniformly reactive, or cause the standard curve to become unusable. Before you repeat your ELISA experiment, read these troubleshooting tips to identify possible sources of your saturated signal error, and solutions to solve it.

ProblemCauseSolution
Substrate problemsExcessive substrateDecrease substrate concentration
Incubation time too longReduce incubation time
Reduce incubation temperature
Substrate activation before useMake substrates immediately before use
Plate errorInsufficient washingFollow manufacturer’s washing instructions
Thoroughly drain plate after washes
Plate sealer not used or reusedCover plate with plate sealer during incubations
Use fresh sealer every time
Plate read at incorrect wavelengthUse recommended wavelength/filter
Check that plate reader is set up for the type of substrate used
Antibody problemsExcessive antibody concentrationReduce antibody concentration
Nonspecific antibody bindingUse a different formulation of coating solution
Use Boster antibodies guaranteed to only react with their targets

Keywords:- ELISA, Troubleshooting, Saturated Signal, O.D., Off Scale