Download Technical Guide for ELISA - Protocols - Troubleshooting

Troubleshooting
Problem
Possible Cause
Solution
High Background
Insufficient Washing
•
•
•
•
Enzyme conjugate at too high a
concentration
• Check dilution. Titrate if necessary.
Insufficient Blocking
• Increase blocking protein concentration.
• Try a different blocking protein.
• Increase blocking time.
Incubation times too long
• Reduce incubation time.
having
Interfering substance in samples
or standards.
• Run appropriate controls.
• Perform “recovery assay” to determine
masking effects.
trouble.
Contaminated buffers
• Make fresh buffers.
Reagents added in incorrect order or
incorrectly prepared
• Repeat assay.
• Check calculations and make new
buffers, standards, etc.
Contamination of enzyme with inhibitor
(azide for HRP or phosphate for AP)
• Use fresh reagents.
Not enough reporter antibody used
• Increase concentration.
Problems with the standard
• Check that standard was handled
according to directions.
• Use new sample.
Capture antibody or analyte did not bind
to plate
• Restandardize coating conditions.
• Increase concentration of coating
component.
• Increase coating time.
• Dilute antibody/analyte in phosphate
buffer to insure that no other protein
is present.
• Change plate type to high binding.
• Try covalent linkage plates.
Buffers contaminated
• Make fresh buffers.
Now
what?
KPL, Inc. • 800-638-3167 • 301-948-7755 • www.kpl.com
I’m
30
7.
No Signal
See washing procedure - page 14.
Add detergent to wash solution.
Increase number of washes.
Add 5 minute soak step between
washes.
• Add protein to the wash solution.