I'm trying to do ELISA for some molecules in human ascites.
Before doing main experiment, I did "spike and recovery test" to examine if there is any matrix effect in my samples.
The recovery rates were less than 10%, which means that there is a lot of matrix effect that can lead to wrong results.
Then, what can I do? Can Western blotting be a suitable replacement to ELISA?
Thank you in advance.
Hi Tae-Shin Kim ,
Are your molecules proteins, hormones...? Western blot will work well for proteins because PAGE will clean your sample. However, if the problem is "molecules extraction of your sample", no method will improve your results since the target was lost during sample preparation.
Best,
Jacó
Have you try troubleshooting your ELISA?
As you know, 'matrix effect' in ELISA occurs when the target antigen interacts with matrix components in plasma or serum samples. In your case, these matrix components can be endogenous biological components like phospholipids, carbohydrates, and metabolites present in the ascite (abnormal fluid buildup in abdomen).
You may obtain weak or noisy results (high background) as the matrix component mentioned could potentially reduce the binding of the antibody to the target protein, or non-specifically bind the antibody during ELISA.
Here, I include some tips to help reducing the 'matrix effect' during ELISA:
1. Centrifuge the sample to separate matrix components from soluble antigens, reducing the concentration of matrix components and their effect on results.
2. Increase dilution to reduce matrix component binding and mitigate the matrix effect.
3. Make sure there is minimal blood components in ascites.
As for western blot, it is used to detect specific protein molecules from among a mixture of proteins (in your case, ascites). Both ELISA and western blot depend on detection of antigens by specific antibodies. In addition, ELISA assays are technically less difficult then Western blots. Therefore, I would suggest you to proceed with troubleshooting your ELISA first before switching to Western blot as the latter will need more optimisation and troubleshooting. Thank you.
I hope this helps you! If anyone has any other better suggestions, please don't hesitate to correct me. Thanks again.
I would also recommend to try Western blot analyses when ELISA is not available for your work.
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