I used ELISA to determine the OD value and of my samples. My diluted samples gives the same OD value. I made a 10x dilution and 100x dilution. Both dilutions gives a OD value of almost 3 and my background is around 0.3. I do not understand why my dilution results in no change of the OD value.
Hard to answer without more info. '3' may represent a saturated reading, in which case you're actual absorbance is above '3' (since a dilution is also '3'). If you performa a serial dilution of the sample down to background ('0.3'), then you can extrapolate the actual absorbance from what the absorbances are of the dilutions in the linear range of that dilution series.
An OD value of 3.0 seems super high . Usually the signal maxes out at around 2.0-3.0 (depending on the reader). It could be that you have too much of your protein even at 100x dilution (this would mean you would have an absorbance value of 3.0 for 100X, 10X dilutions and your undiluted sample). Maybe try diluting your sample to 1000X and 10000X (and more) and see if your signal goes down with dilution.
Your sample type should also be taken into consideration. I do not know what your background sample was (although you said your signal was 0.3, make sure you have the appropriate negative control i.e. use the same sample type as your test sample). Plasma-serum samples could give high non specific signals.
Hello ,
In my opinion your sample is highly concentrate so you must dilute it until you rich an absorbance under 1,5 (x500 , x1000 ...).
You can check the discussion on " Absorbance limits " on ResearchGate:
https://www.researchgate.net/post/Absorbance_limits .
Ridha
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA