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IL-4 ELISA detection problems


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#1 LCameronLab

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Posted 24 January 2012 - 02:14 PM

Hello everyone,

I work as a tech (in training at the moment) at a research lab looking at the CRTh2 gene and it's role in asthma and other inflammatory diseases. At the moment we are running into a few problems in our efforts to optimize some experiments. Please take a look and contribute any advice or thoughts. If you need more info/details please just ask as I will attempt to be brief, and am no expert. I am excited to see what online discussion can bring to the research table!

For now I'm wondering if anyone has any experience working with the IL-4 cytokine. We are having trouble detecting IL-4 in PGD2 treated CD4+ CRTh2+ cell supernatants using ELISAs. At the moment we are using Diaclone ELISA IL-4 Eli-pair Kits and getting very low detections even though standards go down to 1.1pg/ml. Does anyone know any good protocols/kits for IL-4 that have worked well? Any that have accurate detection at very low concentrations? We are trying different dilutions, new serum (we were using FBS and are testing if Human Serum makes a difference). Supernatants are from 2million cells/ml cultures, we are trying 4million cells/ml culture to see if IL-4 production gets bumped well into detectable ranges for our kits.

Please share your thoughts and experiences. We would greatly appreciate your insight.
Sincerely,
BJoseph

#2 PAO_ahac

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Posted 27 January 2012 - 12:32 PM

Ok...you are seeing very low levels of product."getting very low detections"...meaning you don't see very much being produced?

So, you want kit that is more sensitive? much less than 1 pg/ml. If you can't find a kit you can always increase your incubation time/temperature/volume of sample introduced, concentrate your samples before testing.

I would concentrate the sample, retest, and back calculate the concentration.

#3 LCameronLab

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Posted 02 February 2012 - 01:51 PM

Thanks for the reply and your suggestions.
Yes very little IL-4 is produced in some of our samples.
I found some high sensitivity kits that detect down to 0.25pg/ml but they are quite expensive. If anyone is interested the link is here: http://www.ebioscien...y-elisa-kit.htm

We are going to tweak our experiments as you suggested, for incubation time and temperature, and also for different amounts of treatment. We have found that more cells/ml and human serum make small increases (however they are statistically insignificant).
Your suggestion to concentrate is interesting, have you concentrated culture supernatants before? Do you recommend any methods?

Thanks,
Bjoseph

#4 PAO_ahac

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Posted 07 February 2012 - 05:41 AM

Many years ago tested supernatants for production of Mab; same principles apply. You can try Millipore/Merck centricon using MWCutoff 2x smaller than the IL4. If you can pool multiple supernatants of the same condition and concentrate x 100 you should get some decent signals.

Additives as you mentioned above will only play a small role and not increase your sensitivity. If you wish to go down that road then you would have to add PEG to your sample assay buffer to increase interaction/exclusion of water during the test...however this also increases background.

Concentrating sample and increasing sample volume, incubation time/temp would be the first things to examine.

good luck




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