Dear all,
I am trying to measure IL-1beta levels in the ascitic fluid of MSU induced peritonitis mouse model by using Duoset IL1B ELISA(R&D).
However, under all conditions, the absorbance is below the well of zero concentration. For example, the sample (ascitic fluid) has an absorbance of 0.1 when the standard well at concentration 0 has an absorbance of 0.2.
FACS confirms that we are able to induce inflammation. The calibration curve has been measured without problems.
A peritoneal lavage was conducted with cold PBS and cellular components were removed by centrifuge before use.
Ascites fluid is administered in its undiluted form, but should we assume that there is some substance in it that inhibits the reaction?
Is there any solution I can do?
Thanks,
Hello,
To measure IL1B (and IL1A) I always had to concentrate my samples. I used
Amicon Ultra-15 3kDa (to keep as much as proteins as possible), and concentrated the samples by centrifugation (4000 g for 10 min, but you need to adapt the time according to the concentration you need). It works fine with the DuoSet.
I agree with Dr. Christine Pich-Bavastro the PBS dilutes the samples too much. Besides the concentration column, you can use a vacuum spin at 4C if you have access to it or put samples (with open lids) into the vacuum chamber with silica gel overnight at 4C. You will need to add a protease inhibitor cocktail for prolonged work with ascitic fluid samples at 4C. Alternatively, you can try WB of the ascetic fluid as it is more sensitive than ELISA, but you will have a headache with the housekeeping protein. Reviewers usually do not like the whole protein staining such as Coomassie.
Dear Dr. Christine Pich-Bavastro and Dr. Anton Lennikov
Thank you for your kind advice! I will try again by using Amicon Ultra-15 3kDa.
Thank you very much.
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