14 replies to this topic

[Mati]

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 ??


Thanks

[almost a doctor]

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.

Edited by almost a doctor, 16 April 2012 - 02:17 AM.

[Kat T]

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?

[Ben Lomond]

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

[Mati]

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

[BioMiha]

Do you have serum (containing IgG) in your cell medium?

[Mati]

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

[BioMiha]

That does not answer my question. Do you add serum as a supplement to the cell culture medium?

[Mati]

Yes usually i add FBS in my medium ? Is this because of FBS?

[almost a doctor]

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.

[Mati]

Thanks for your kind Suggestion
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??

[Kat T]

You may not be able to grow your cells without FBS in your medium.  I have run similar assays previously and reduced the level of FBS to 1%, which has successfully lowered the background level in the ELISA and allowed me to detect the response by the cells.

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.

Good luck!

[Ben Lomond]

If your capture and detection reagents have not been selected to have demonstrated clack of cross reactivity to bovine IgG, then yes, you will get the non-specific results you describe.

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.

good luck!

[PAO_ahac]

Just an offside question...with cell culture can an IgG stripped serum be used as a supplement to grow cells?

[Ben Lomond]

Bovine IgG doesn't help cells grow, and Gibco, Thermo and others sell 'ultra low' IgG fetal bovine serum, which they claim is just as good.  Looking at the specs, it still has a few micrograms per mL of bovine IgG in it, so for this application, a stripped serum might be preferable if the process can remove all the IgG. Protein A and G won't bind all subclasses, so it would have to be passed through some sort of anti-pan bovine IgG affinity matrix. Conditioning the cells to serum free would be simpler.