Elisa using HEK culture medium
Posted 16 April 2012 - 01:46 AM
I tried with more washing and blocking but got same results.
My negative and positive control looks ok what should i will do to improve my Elisa system ??
Posted 16 April 2012 - 02:17 AM
A lot more detail of what you are doing will help us help you.
Edited by almost a doctor, 16 April 2012 - 02:17 AM.
Posted 16 April 2012 - 01:13 PM
Posted 16 April 2012 - 03:00 PM
Posted 16 April 2012 - 07:38 PM
I am looking for IgG in cells supernatant, usually the quantity of IgG is not so much in culture supernatant and not all samples can produce IgG successfully.
I coat antihuman IgG overnight then use cell supernatant as a primary A.B .
Seconadry A.B is antihuman IgG ALP.
3% Bsa as blocking reagent.
I used Human IgG as Positive control.
Negative control , only coating and secondary A.B no IgG. My negative control gives me no result showing that washing steps and blocking steps are performed good and no cross reactivity.
I dont know why all supernatant gives me result, because the chance of IgG detection is very less, i believed only one or two samples should be positive not all.
Thanks for any suggestions
Posted 16 April 2012 - 11:49 PM
Posted 16 April 2012 - 11:50 PM
Posted 17 April 2012 - 12:36 AM
Posted 17 April 2012 - 12:40 AM
The negative control you are currently using (no sample) as you said shows your washing and blocking are good, but it doesn't give you the actual background level. You need to have a blank that consist of the same type of sample that you are testing, but negative (this can even be the media you use to grow the cells, not necesarily supernatant, although supernatant from "negative" cells will be better to account for any other cell-derived component interfering with your assay).
Hope this helps.
Posted 17 April 2012 - 02:11 AM
I dont have blank or negative control supernatant but i will try using this system now.
What about using medium with FBS and medium without FBS and compare results by Elisa??
Posted 17 April 2012 - 01:16 PM
You should definately run "growth media only" as one of your samples to determine how much of your activity is due to the media that the cells grow in. Just take an aliquot out of your bottle at the same time as you collect your cell supernatant and treat it in the same way as you do for the rest of your samples (i.e. if you freeze your samples, then you should freeze your media too). It is likely that your high background is due to your FBS.
Posted 18 April 2012 - 05:22 PM
consider using a capture reagent that has been preabsorbed against bovine IgG or one that is proven not to cross react with bovine immunoglobulins.
antibodies to consider might include southern biotech 2081-01 or jackson 109-005-088 but browse through their websites and there are many options.
The detection reagent isn't so important, as the serum is washed off before you apply that reagent. Southern biotech 9040-04 is an excellent Fc gamma specific mono that would be a good detector for an anti-Fab specific or H+L (heavy and light chain) bovine preabsorbed poly capture reagent.
Posted 19 April 2012 - 02:22 AM
Posted 19 April 2012 - 05:32 PM