ELISA for DNA/RNA?
BIOLEV at UNIPAD.UNIPD.IT
Thu Feb 21 06:56:00 EST 1991
>Has anybody come up with a way of fixing DNA or RNA to the bottoms of
>microtiter plate wells? Say, for example you wanted to fix total RNA
>samples from a large number of tissue samples (eg. a timecourse
>experiment), and then hybridize with a biotinylated probe. Next add
>streptavidin-conjugated alkaline phosphatase, and substrate, and read the
>result in your microtiter plate reader. Essentially we're talking about
>an ELISA for DNA.
> | %%%%% | streptavidin-conjugated enzyme
> \ ---- / biotinylated antisense RNA probe
> \ ^^^^ / unlabeled total RNA (fixed to plate)
>This is such an obvious idea that I would think there must be a kit
>somewhere for it, but I don't recall having seen it in the literature.
Yes, well I actually am using such a type of assay to detect Benzo(a)pyrene
DNA adducts using an ELISA assay with a policlonal antibody raised against
the BP-DNA adducts. The DNA samples are fixed to the bottom of the microtiter
plate by first heat-denaturing them and then placing the solution in the wells
and allowing the wells to dry out overnight at 37 C. The DNA then binds to the
wells fairly well (say 100 ng DNA/well). Then incubate the first antibody with
the DNA containing wells and after this incubate a second antirabbit-alkaline
phosphatase conjugate and then use either a fluorochrome (methylumbelliferyl)
or PNPP for visible EIA readers (405 nm). This is just a quick run through the
method. If any further details are required......Glad to be of help!!!!!
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