Just to rehash the process, the PA binds to the ATR and is cleaved by the furin-like protease leaving the PA 63kDa fragment bound to the cell surface where it eventually forms the heptamer and is endocytosed along with either the EF or LF. The PA 20 kDa fragment is said to "dissociate to the medium." But where does it go? Is it opsonized and phagocytosed? Does it have a role in limiting the immune response to bacterial infection? ELISA assays have been developed to detect antibodies to the lethal and edema factors, but with B. anthracis' penchant for limiting the immune response, AB titers to the factors may not rise to a detectable level until late in the infection. Additionally, since the PA 63 fragment and both of the factors become endocytosed, AB may not find them until late in the infection. My proposal is to develop monoclonal antibodies to the PA 20 fragment to be used in an ELISA assay, which may allow earlier detection of B. anthracis infection through use of this this extracellular fragment from a serum sample.

2) Cleave the PA by incubating with soluble furin at 37 C. Furin will cleave PA at the RKKR site yielding a 63 kDa fragment and a 20 kDa fragment which can be visualized on western blot using anti-PA polyclonal rabbit serum. A PA mutant at the RKKR site can be used to ensure furin is cleaving at the known site.

3) To ensure that you have produced biologically active cleavage fragments. Isolate the 63 kDa fragment from the gel and incubate along with recombinant lethal factor using the macrophage assay to test for cell lysis.

4) Isolate the PA 20 fragment from the gel and inject into a rabbit. Isolate B-cells, fuse to a myeloma cell and grow on HAT medium to select fused cells. Initiate production of a second AB against a different domain of the fragment by cleaving the fragment with an endonuclease, isolating a fragment and inject in a rabbit. Ensure that the first AB does not cross react with this domain but that this AB will react to the PA20 fragment.

5) Produce a sandwich ELISA by adhering the first AB to a microplate well. Add the PA 20 fragment and wash away any unbound. Add the second AB with enzyme label to the well and wash away any unbound. Add substrate which will produce a color change when cleaved by the attached enzyme. Test and retest to ensure that the AB's are specific to PA20 and are sensitive enough to detect it from serum samples. So…make sure they don't cross react with other similar bacterial antigens or proteins from a serum sample.