Hi Juan Carlos, The IL-1b and IL-6 levels in normal patients are very low.  Check your assay with serum from patients with an infection.  These should be high.

Concentrating your samples by dehydration-evaporation? Reducing their volume thereby increasing solute density? Special caution on how not to degrade any cytokines present. Some antibodies (but some not) also detect denatured proteins or protein fragments. 

Hi Juan,

Have you optimized your blocking and Ab incubation times to reduce background and enhance binding respectively? You may want to move a more sensitive method - I have used the BD biosciences Flex kit multiplex and got nice results - sensitive to fg levels. The luminex platform is also nice - it is even more sensitive I believe.

Good Luck!

Hi Juan, to follow on from Edmund's suggestion,  you could spike a couple of your samples with a known amount of IL-6 or  IL-1b to double check the sensitivity of your assay in the background of the actual patient plasma. 

Were the patient samples processed and stored optimally?  How long have they been frozen and at what temperature? All of  these factors have a bearing on the stability of the cytokines.

Increase EIA sensitivity by increasing incubation times and temperature.

Thank's a lot for your comments.

Edmund Pool: I already try serum/plasma from Dengue infected patients...

Rosemary L Sparrow: the samples were well processed and store for less than a month at -80ºC.

Falk Fish: thank's I will try this.

Best

try acetone precipitation ...

Hi Juan

Try to assay fresh samples.  Are you near a hospital with a pathology lab?  If so try fresh serum from infected patients. This will indicate if your current samples were degraded upon storage. For IL-6 and IL-1b sera from patients infected with bacterium should work well.

Are you using a specific kit for human cytokines? Kits for other species often do not detect the human proteins.

Did you freeze at -80 C or lower? If not samples will probably be degraded.