Follow our Enzyme-linked immunosorbent assay (ELISA) troubleshooting guide to quickly identify and get solutions for common protocol issues.

Identify the problem with your ELISA test results from the options below:

Poor Standard Curve

High coefficient of variation (CV)

High Background

Sample readings are too high

Sample readings are too low

Note: If using a StressXpress® ELISA assay kit, please reference the StressXpress® ELISA troubleshooting tips below. If you have created your own ELISA assay kit, please reference the other ELISA troubleshooting tips.

Poor standard Curve

Standard improperly prepared:

• StressXpress® – Confirm dilutions were calculated and conducted correctly
• StressXpress® – Spin down standard before opening vial
• If reconstituting a standard from a lyophilized powder, ensure all material is dissolved before dilution
• Use calibrated pipettes and follow proper pipetting techniques

Standard degraded:

• StressXpress® – Ensure standard is stored as directed to avoid degradation
• Use a fresh standard

Curve does not fit:

• Use a different scale or curve fit

Pipetting errors:

• Ensure multichannel pipettes are calibrated

 

High coefficient of variation (CV)

Well bottom scrapped:

• Avoid contact with the bottom of the well during pipetting. Aim the pipette tip to the side of the well to avoid disrupting the bottom

Incorrect volumes added to wells:

• StressXpress® – Follow the protocol for specific volume of reagents added to each well
• If using an automatic washing system, ensure all the ports are clean and unobstructed

Edge effect:

• Ensure the plate temperature is consistent across the plate, and that all reagents are at room temperature

Pipetting errors:

• Ensure multichannel pipettes are calibrated

Washing errors:

• Ensure all buffers are fully removed from all wells between wash steps

Non-homogenous samples:

• Ensure samples are homogenous before use by mixing thoroughly
• If necessary, spin down any particulate that will stay in solution

Inadequate shaking:

• Ensure the plate is placed on a shaker during all incubation steps

Inter-well contamination:

• Avoid touching pipette tips to wells or reagents in wells
• If reusing the same plate sealer between incubations, ensure that reagents have not come into contact with the sealer

 

High Background

Insufficient washing:

• StressXpress® – Follow the protocol for correct volume and number of washes
• Increase the volume and number of washes prior to addition of the substrate solution

Evaporation of sample:

• Tightly cover plate with provided cover or parafilm

Blanks incorrect:

• StressXpress® – Follow the protocol to correctly combine reagents for the blank wells
• Ensure the blank values have been subtracted from all other readings

Reaction was not stopped:

• StressXpress® – Ensure the correct volume of stop solution was added to wells
• Use fresh stop solution

TMB exposed to light:

• Store TMB in a dark vial prior to use

TMB contaminated:

• Ensure TMB is clear and colorless prior to addition to wells

Blank wells contaminated:

• Avoid touching multichannel pipette tips to wells or reagents on the plates

Plate bottom is dirty:

• Thoroughly clean the bottom of the plate

 

Sample readings are too high

Samples too concentrated:

• StressXpress® – Dilute samples as outlined in the protocol or optimize for your particular samples
• Increase the dilution of the samples

HRP concentration incorrect:

• StressXpress® – Ensure that HRP was used at the appropriate dilution outlined in the protocol

Incubation time too long:

• StressXpress® – Incubate samples as outlined in the protocol
• Decrease the incubation time

 

Sample readings are too low

Antigen levels too low:

• StressXpress® – Dilute samples as outlined in the protocol or optimize for your particular sample type
• Decrease the dilution of the samples or concentrate samples

Development time too short:

• StressXpress® – Follow the protocol for the optimal development time
• Increase development time appropriately

Incubation time too short:

• StressXpress® – Incubate samples as outlined in the protocol
• Increase the incubation time

Incubation temperature too low:

• StressXpress® – Incubate samples at the temperature outlined in the protocol
• Optimize incubation temperature

Plate is expired:

• StressXpress® – Check the expiry date of the kit located on the side of the kit box
• Coat a new plate

Antibody concentration too low:

• StressXpress® – Use the antibody at the concentration outlined in the protocol
• Use a higher concentration of antibody

Substrates not at RT:

• All reagents must be at room temperature (25⁰C) before use

Incorrect wavelength:

• Ensure the plate was read at the appropriate wavelength

Delay in reading plate:

• Plates should be read immediately after the addition of the stop solution

Incompatible samples:

• StressXpress® – Ensure your species and sample type have been validated for use with the kit

Over washing:

• StressXpress® – Follow the protocol for appropriate volumes and number of washes
• Pipette wash buffers gently into the wells
• If using an automatic washing system, ensure the pressure is correct

Wells dried out:

• Do not let wells dry out during the protocol