Antibody Guarantee - ELISA

Please select the link that most accurately describes the problem you observe:
	No signal
	Very weak signal
	High background
	Poor standard curve and duplicates

No signal

Possible causes:

• Has a step been omitted by mistake? Repeat ELISA following the protocol carefully
• Is it the right substrate for the right enzyme? Double-check
• Is there an enzyme inhibitor present in the system, e.g. sodium azide inhibits peroxidase activity? Make sure system does not contain an enzyme inhibitor
• Reagents too old? Do not use reagents if past expiration date
• Have reagents been stored incorrectly? Check storage instructions

Very weak signal

Possible causes:

• Was the incubation time adequate? Check recommendations
• Was the incubation temperature right? Check recommendations
• Conjugate concentration too low? Check recommendations for antibody and use a higher concentration
• Wrong filter in ELISA reader used? Use correct wavelength setting
• Is there an enzyme inhibitor present in the system, e.g. sodium azide inhibits peroxidase activity? Make sure system does not contain an enzyme inhibitor
• Reagents too old? Do not use reagents if past expiration date
• Have reagents been stored incorrectly? Check storage instructions

High background

Possible causes:

• Plate not sufficiently washed? Make sure all the wells are filled with buffer during washing step
• Conjugate concentration too high? Check recommendations for antibody
• Non-specific binding of antibodies? Try different blocking conditions
• Cross-reaction of the detection antibody with coating antibody? Perform a control with only the two antibodies without sample between
• Reagents have expired? Do not use reagents if past expiration date

Poor standard curve and duplicates

Possible causes:

• Inefficient or improper washing? Make sure all the wells are filled with buffer during washing step, wash enough but not too hard
• Wells are not aspired adequately? Ensure wells appear to be dry after aspiration
• Poor mixing of samples? Mix adequately and equilibrate to proper temperature
• Pipetting error? Check pipets and pipetting technique
• Plates not clean? Plates should be cleaned on bottom before measuring absorbance

If the problem you are observing is not accurately described or remedied by any of the above suggestions please let us know using this form

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