Pathogenic organisms are believed to be responsible for a number of infections, neurodegenerative and as well as some types of neuropsychiatric diseases. Detection of neuroactive pathogens, at the earliest stages, would be a critical factor in the diagnosis and treatment of any possible neuropathological consequences. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are already being used separately as specific microbial detection methods. In the present project, we propose to develop a technology of pathogen detection, which combines the advantages of ELISA and those of PCR. This is expected to result in a highly efficient, reproducible method for the detection of very small numbers of pathogens, even before they can elicit the normal host immune response. The project will be carried out in two phases. In phase 1, the method of immuno-PCR will be standardized by measuring proteins and peptides that have special importance in brain function or pathology. This will be done using either brain cells where they are specifically and abundantly expressed, or in heterologous cells, such as lymphocytes, where they are expressed at very low levels, by a phenomenon known as """"""""'illegitimate transcription"""""""". In phase 2 we will extend the protocols developed in phase 1 to detect and quantitatively measure some of the pathogens that are commonly found in human populations and which are of great concern to public health authorities. Four different approaches to immuno-PCR will be compared, based (a) on electrophoresis and ethidium bromide staining of the amplified DNA and densitometry of the band obtained: (b) measurement of fluorescence produced following reaction of the PCR mixture with the dye, PicoGreen; colorimetry following reaction of chromogenic substrate with digoxigenin-alkaline phosphate ligated to DNA; and (d) fluorometry of the complex produced by the action of a fluorescent substance (AttoPhos) on the DNA-digoxigenin complex.
(a) Development of rapid and highly sensitive, early detection kits for neuropathogens in human and animal tissues and body fluids; (b) Evaluation of the efficiency of vaccines, commonly used in persons at risk for infection; (c) Evaluation of the immune reaction subsequent to primary infection or immunization, and (d) possible identification and treatment of microorganisms responsible for idiopathic neuropathogenesis.
