hIL-4 Immuno & ELISA assay

M. Doherty M_Doherty at NIH.gov
Tue Apr 23 08:52:53 EST 1996

In article <317BD542.63AD at aix1.danadata.dk>, Jens Rainer og Birgit
Sehested Hansen <jrhbsh at aix1.danadata.dk> wrote:

> Hi I am a new user here so I dont no if it the right way to use the net,
but I try anyway. 
> I have stimulatet humane lymphocytes using PHA for 24, 48 and 72 hours
to get the cells to secrete IL-4. But wery low levels (100-300 pg/ml) were
> with a sandwich  ELISA. The assay is performed in microtiterplates at a 
> cellconc. of 1e6 cells/ml.
> There are two problems 1) to get the ELISA test more sensitive or to get more 
> hIL-4 producet. The purpose of the assay is to test new compounds inhibiting 
> effect of the IL-4 production, and that is difficult now, concerning the low 
> IL-4 production and the low senitivity.
> Do you have experiense, do I try with other stimulators etc.

OK, several suggestions.
First you can safely increase your number of cells to 4 million cells/ml
under these conditions.  
Second, I would try either Plate-bound antiCD3 (2 micrograms/ml) or ConA
stimulation (2.5 micrograms/ml)
Third, is the type of feeder cells.  If you use T-depleted spleen, you
will generally get better IL-4 yields than with macrophages or T depleted
peritoneal exudates.

Finally, if all of these are not sufficient, try using a bioassay (growth
of CT4S cells, for example) to measure IL-4.  In my hands, this has been
more sensitive than ELISA.

Good Luck,
Cheers Mark

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