I want to perform an ELISA with dsDNA by capturing the biotinylated dsDNA on neutravidin. Then we detect binding antibodies. This method is not really reliable in our hands. Has somebody have experience with this in terms of plates, buffers, salt .... Does anybody know the pitfalls that I have to avoid?
How long are the DNA molecules? If they are very short oligos, they can dissociate into single-stranded DNA when the plate is washed repeatedly.
Thanks Adam for your advice. The number of bp are around 18. Do you think this long enough? Which length would you suggest?
What plates are you using? I had lots of problems with commercial coated plates, until I switched to coating my own plates with streptavidin I had huge problems with variation.
The stability of a short oligo of 18 bp to dissociation depends on the GC content, with higher GC:AT ratio giving greater stability.
Alison, we use Nunc plates and coat them with Neutravidin. Adam, thanks for your help. Do you think that it is usefull to add a non-sense sequence with a high GC:AT content, which size would you give to a sequence? (I am thinking about GS linkers in proteins)
Doing what you suggest would be a good way to find out whether dissociation of the strands is causing a problem.
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