We previously reported on the development of a 1-dimensional gel electrophoresis and Western immunoblot analysis for use in the detection of 14-3-3 family of proteins in the cerebrospinal fluid of patients with spongiform encephalopathy. We early recognized that the Western blot procedure was a quantitative assay giving either a positive or a negative reaction and although the results obtained in the testing of over 1500 specimens has provided for a sensitive and specific test for the spongiform encephalopathies the sensitivity is limited by the assay itself. We thus worked on the development of a potentially more sensitive test, a quantitative test that would be more informative in the preclinical/asymptomatic incubation stage of the diseases in both humans and animals. During the period covered by this report we have developed an ELISA test for detecting the quantitative analysis of 14-3-3 proteins. Seven different polyclonal antibodies and two monocloncal antibodies to 14-3-3 protein have been developed, standards have been obtained. A double sandwich has been developed and standardized. The ELISA test is sufficiently sensitive (<2.1 ng/ml) and the between-day precision is acceptable (cv=0.10). The plot of the standard curve is linear in the range of 2.1 to 135 ng/ml with a regression correlation coefficient at unity (R2=1:000).
