Hi,
I am a newbie with Hybridoma fusion and selection via ELISA and would be greatful for some tips for a screening problem I have.
I completed the fusion successfully last week and there were colonies growing in a lot of the wells. I started the screening yesterday with the first 100 clones via ELISA for my target protein.
I have done this ELISA multiple times with mouse sera and it works fine- no background etc. But in screening my hybridoma supernatants i got a signal in all of the wells... I block with 5% FCS overnight at 4°C (minimum 16 hours). I guess I have a high concentration of antibodies from the supernatant binding the plate, but I don't know what would be the best option to stop this considering blocking is already quite long?
There were about 3 wells that were particularly positive in the ELISA and I decided to expand these in the hopes these were positive above the background of the plate. However, when i check these wells, there are clearly other adherent fusiform cells/ contamination? growing in these in all 3 wells (see attached pics). Can anyone suggest what this contamination is? and why it would give such a strong signal in the ELISA?
Thanks in advance
Hannah
More information on the ELISA needed really.
What are the differences in the procedure, if any, when you test mouse sera versus hybridoma supernatants? Did you do a pre-immune mouse serum control?
Presumably you have an anti-mouse HRP or AP conjugate? If so, the issue may be that you have bovine Ig in the FCS and that your anti-mouse enzyme conjugate cross reacts with bovine IgG.
It is far more usual and cheaper to block plates with BSA (1%) and you certainly do not need an overnight incubation. 1h should be sufficient. This approach to blocking removes any potential issues with bovine Ig in FCS.
Should you still have backgrounds after a BSA block, then cross-reaction of secondaries can be ruled out and, instead, the issue is more likely to be inadequate washing of the plate prior to addition of enzyme substrate. However, this would be true both for mouse sera and for hybridoma supernatants, which is why you should consider if there are any assay differences for the two sample types.
Another thought - do you have any wells where you omit supernatant or, preferably, add just unused tissue culture medium? If not, then I suppose you may have an improbable situation that all wells have secreting cultures. However, overall it feels more like an assay wash problem or a cross reaction of the secondary.
Thanks for the suggestions Nick.
There are no differences between the sera and supernatant assays, all antibodies/development is exactly the same. I wash with 0.05% Tween in PBS for at least 1 minute (soaking), 4 times between steps and 6 times between the addition of supernatant and the secondary. Pre immune sera and naive mouse sera is negative.
I have tried with just supernatant from myeloma cells and this well is negative so I am unsure whether it is an issue with the secondary/FCS. Neverthless I am trying a BSA block now and will increase washing before addition of substrate to see if things improve.
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