I performed one ELISA trying to use IL-10 KO supernatant samples as negative control. It came out they gave positive for IL-10. The same result as for WT mice... How can I resolve the issue?
I would personaly suggest to make a look at once again on your antibodies because your ELISA sensitivity and specificity is highly determined by the affinity of your antibody to your cytokine/antigen. moreover, some antibodies might have a heterophilic nature and there is a tendency of non specific binding and gives you false positive result which is probably the same in your case. good luck.
Hi, Ana.
I suggest to try to find optimum dilution of the fluid to be analyzed by giving +ve and -ve in WT and KO, respectively.
Make sure your fluid is free of any particles, microbes or microscopic fragmented cell debris. Spinning down at 12,000 rpm for 10min, not less!!!
If it wouldn't work. use primary antibody conjugated to biotin, followed by enzyme conjugated to streptavidin. This system allow more narrow specificity. People used rat anti-IL-10 ab for detection of mouse IL-10, too. Thus you have more choices.
Hope, solve the trouble.
If you bought the ELISA as a kit I'd be speaking to the manufacturer as it is clearly non-specific.
If your own creation, as above you need to look at the Ab combination
murray
I guess not at all.
IL10 ko mice still express a truncated form of IL10 that is detected in ELISA.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA