Chris Jones (Chris.Jones at nottingham.ac.uk) wrote:
: Please excuse my ignorance but could someone tell me what are the differences
: (by which I mean sensitivity, specificity etc. rather than the mechanics)
: between developing an ELISA vs. RIA. I have noted that there appear to be more
: commercial kits for RIA available, certainly from Peninsula, but my lab would
: prefer to avoid Radioisotopes. If there is an RIA kit commercially available
: is it possible to pocket the serum and develop an ELISA?
: I have recently been supplied with a rabbit polyclonal serum and need
: to set up a quantitative assay from scratch. Would it be as easy to set up an
: ELISA than an RIA.
: Excuse my ignorance again I am just a poor molecular biologist!
It is possible to set up either assay, however the sensitivity is usually
greater in RIA vs. ELISA. If you only have a single antibody then a
competetive RIA with labeled antigen would be the easiest to perform from
scratch. Alternatively, you can split your antibody and biotinylate half
(to increase the signal) and perform a sandwich ELISA. This may not work
if all of the available binding sites are used up in the first binding
step. The specificity is entirely dependent upon the antibodies used,
affinity, and cross reactivity with other antigens. A monoclonal antibody
(or pair from two surces ie. mouse and rat) would provide the highest
specificity. The other key factor is the quality of your standards. Good
luck!
--
Richard Borschel
borschel at netcom.com
