Elisa using HEK culture medium - (Apr/16/2012 )
Hi i am doing Elisa using HEK cells supernatant , i always found the same result or highbackground for all the supernatant samples.
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 ??
If your positive and negative controls work fine, it could be either that your cells are not expressing (or secreting) the protein you are trying to detect. Or that the levels are too low for your assay limit of detection.
A lot more detail of what you are doing will help us help you.
Do you run your negative controls with the HEK media? Is it the media that is giving high background results or is it only when the cells are present?
Please give more specifics relating to the ELISA you are running: analyte, antibodies, substrate, blockers and diluent. What is incuded and what is not included in your control wells. Without this information, any advice you receive will be generic
Thankyou all for your concern here are the details
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
Do you have serum (containing IgG) in your cell medium?
If my transfection was successful then only some cell can produce IgG not all .So i want to detect only positive cell medium by Elisa
That does not answer my question. Do you add serum as a supplement to the cell culture medium?
Yes usually i add FBS in my medium ? Is this because of FBS?
I agree with BioMiha. If your supernatats have FCS/FBS the IgG present in this will hinder your results. Do you have a blank or negative control sample, ie: supernatant without IgG = cells that will definitely NOT express IgG (non-transfected?), if so, how does this one look?
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.