Improper standard dilution

Confirm dilutions made correctly
Standard improperly reconstituted

Briefly spin vial before opening; thoroughly resuspend powder
Standard degraded

Store sample as recommended
Curve doesn't fit scale

Try plotting using different scale

Target protein not expressed in sample used/ Low level of target protein expression in sample used
Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay. Ensure you are using a positive control within the detection range of the assay.

Insufficient antibody
Check the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.

Substrate solutions not fresh or combined incorrectly
Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed at the correct concentration.

Reagents not fresh or not at the correct pH
Ensure reagents have been prepared correctly and are in date.

Incubation time not long enough
Ensure you are incubating the antibody for the recommended amount of time, if an incubation time is suggested. The incubation time may require increasing for optimization of results.

Incubation temperature too low
Antibodies will have optimum binding activity at the correct temperature. Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working. Incubation temperature may require some optimization. Ensure all reagents are at room temperature before proceeding.

Stop solution not added
Addition of stop solution increases the intensity of color reaction and stabilizes the final color reaction.

Wells are insufficiently washed
Wash wells as per protocol recommendations

Contaminated wash buffer
Make fresh wash buffer

Conjugate to strong or left on too long
Check dilution of conjugate, use it at the recommended dilution. Stop the reaction using stop buffer as soon as the plate has developed enough for absorbance readings.
Substrate solution or stop solution is not fresh
Use fresh substrate solution. Substrate should be clear (if it has gone blue, this is a sign of contamination and it should be replaced).

Reaction not stopped
Color will keep developing if the substrate reaction is not stopped.

Plate left too long before reading on the plate reader
Color will keep developing (though at a slower rate if stop solution has been added).

Contaminants from laboratory glassware
Ensure reagents are fresh and prepared in clean glassware. Sterilize glassware beforehand if possible.

Substrate incubation carried out in the light
Substrate incubation should be carried out in the dark.

Incubation temperature too high
Antibodies will have optimum binding activity at the correct temperature. Ensure the incubations are carried out at the correct temperature and that incubators are set at the correct temperature and working. Incubation temperature may require some optimization.

Target protein not expressed in sample used/ Low level of target protein expression in sample used
Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay.

Reagents not fresh or not at the correct pH
Ensure reagents have been prepared correctly and are in date

The concentration of samples or positive control is too high and out of range for the sensitivity of the assay.