Immunodiagnosis of prion disease.

The immunodiagnosis of prion diseases is of critical importance due to the transmissibility of these conditions and their fatal prognosis. A panel of monoclonal and polyclonal antibodies have been generated for use in the study and diagnosis of these diseases. This manuscript describes the generation and characterization of these antibodies as well as their diagnostic application.

The role of antibodies to PrP in the diagnosis of transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE) are progressive degenerative disorders of the central nervous system. Efficient and accurate identification of these disorders is necessitated by their transmissibility and fatal prognosis. The availability of polyclonal and monoclonal antibodies to a TSE disease-specific protein marker PrPSC affords the sensitivity and specificity for immuno-diagnostic assays. The majority of PrPSC antigenic sites are species-directed, involve non-self sites and are common to both the normal host precursor (PrPC) and the modified disease form. The availability of these antigenic sites is highly restricted by conformational influences resulting in epitope-dependent restrictions on antibody binding. Diagnostic immunoassays for TSE have relied largely on immunocytochemistry and immunoblotting. Restrictions on epitope availability have lead to the formulation by several laboratories of a variety of techniques to unmask PrP specific epitopes. In addition, diagnosis requires the ability to detect PrPSC specifically in tissue which can also contain immuno-reactive PrPC. Immuno-detection techniques are discussed relative to their range of application, ease of interpretation, specificity and sensitivity.

Brain regional distribution of prion protein PrP27-30 in mice stereotaxically microinjected with different strains of scrapie.

Stereotaxic inoculation was used to examine the role of scrapie agent strain, inoculum, and injection site on the brain regional distribution of the prion protein, PrP27-30. Neither the type of inoculum nor the injection site influenced the distribution of PrP27-30 in brains of mice. Among the parameters examined, only the strain of agent affected the pattern of distribution and the yield of PrP27-30. Although mice injected into the cerebellum had the shortest incubation period, the cerebellum gave the lowest yield of the PrP27-30 among the seven brain regions examined. The positive correlation between PrP27-30 regional distribution and lesion profile (degree of vacuolation) reinforces the role of the PrPSC protein in scrapie pathogenesis.

Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.

Functional consequences of prenatal exposure to lead in immature rats.

Long-Evans dams were given 0.5% lead acetate as their sole drinking solution two weeks before and throughout pregnancy. Their offspring were transferred to normal surrogate dams on the second day after birth. From days 5 through 30, the rat pups were observed for the appearance of developmental landmarks and given behavioral tests (surface righting, negative geotaxis, eye opening, left-right position discrimination and reversal, ambulation and head dipping). Pups of pair-fed-and-watered as well as normal control dams were also transferred to surrogates and received the same tests. Although the lead-exposed rat pups had markedly elevated blood and brain lead on the day of birth (which were still significantly elevated on day 16), they showed no delay, impairment, or any other change on the various functional measures.

Experimental maternal phenylketonuria: an examination of two animal models.

Two satisfactory rat models of maternal phenylketonuria (PKU) have been developed. Continuous subcutaneous infusion into pregnant rats from the 9th-20th day of gestation of either (1) phenylacetate (PA), to elevate plasma levels of unconjugated PA to 0.25-0.60 mumol/ml, or (2) a nontoxic dose (0.2 mumol/g/day) of p-chlorophenylalanine (pClPhe) with L-phenylalanine (Phe), to elevate plasma Phe levels at least 10-fold (1.7-2.3 mumol/ml) and unconjugated PA to at least 0.2 mumol/ml, produced the syndrome of untreated maternal PKU: spontaneous abortion, mortality rate greater than normal among the newborn, retarded growth of fetal body and brain, and a learning deficit among the progeny. From the plasma of rats infused with only pClPhe, a metabolite was isolated and identified as p-chlorophenylacetic acid. This compound, at a concentration greater than 0.15 mumol/ml plasma was found to retard fetal body and brain growth. In the pregnant rat, plasma levels of unconjugated PA, in the range observed in some PKU individuals on a normal diet, effectively induced a simulation of maternal PKU. The results of this investigation support our contention that PA, which is produced in excessive amounts in clinical PKU, is the primary cause of the brain dysfunction.