Recently we evaluate the serum IL-6 level in patients after radiotherapy . Unfortunately 4 of 7 samples are negative.
we used the thermo fisher ELISA kit for this aim and 100 microliter of serum was used as a sample.
Can i overcome this problem by increasing the incubation time to over night?
If your samples are not expressing detectable levels of IL-6, overnight incubation might not help. Longer incubation might give higher ODs (>detectable levels of IL-6) but your background will also increase.
Serum samples don’t express a lot of cytokines under basal conditions and from the little research I did online, IL-6 levels post-radiotherapy are low (See links below). It would also vary depending on the type of disease and also the health status of the patient. You could try adding more sample and see if that helps. You may have better luck looking at alternate cytokines/molecules that are expressed at higher levels.
http://tcr.amegroups.com/article/view/18283/html
Article Effect of radiotherapy on serum level of interleukin 6 in pa...
Agree with Julie Joseph , overnight incubation might not help.
Level of IL-6 depend on the condition of patients. May be your patients is not homogen condition and also the time of sampling can be critical issue
You can find a reference in:
British Journal of Cancer (2004) 90, 2312 – 2316
"Baseline serum levels of cytokines in controls and prostate cancer patients
Both IL-6 and TNF-a were detectable in all control subjects and prostate cancer patients. In all of the prostate cancer patients, IL-6 and TNF-a levels were 5.676.7 and 4.373.6 pg ml1 , respectively,
and significantly higher than the controls (1.170.6 and 1.270.4 pg ml1 , respectively, P ¼ 0.031 and 0.003)..."
Yes, you can. You are only limited by the volume that your wells can hold. Just make sure you are blocking your wells with volumes bigger than your sample volume (i.e. 250-300 uL blocking buffer in this case).
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