Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

Ceruloplasmin (Cp) was isolated from fresh equine plasma by precipitation, cellulose chromatography, and improved ion-exchange chromatography. Purified equine Cp is a glycoprotein having a molecular weight of approximately 115,000. In electrophoresis, equine Cp migrated to the alpha 1-globulin region, its isoelectric point was about 4.15 and consisted of about 890 amino acid residues. Serum Cp concentration was measured by use of the single radial immunodiffusion method. In clinically normal horses, the mean (+/- SD) serum Cp concentration of newborn foals was 2.87 +/- 0.40 mg/ml and that of 3-month-old foals was 5.02 +/- 0.92 mg/ml, which was similar to the adult value. It reached a peak of 6.06 +/- 0.74 mg/ml in 2-year-old horses. The Cp concentration in mares was not statistically different for the perinatal period, but it decreased immediately before and after delivery. Concentration of Cp increased at 6 days after IM administration of turpentine oil, castration, or jejunojejunostomy in adult horses, and increased to peak values twice as high as baseline values at 7 to 14 days, returning to baseline values at 28 days after treatment. We concluded that equine serum Cp is an acute-phase reactive protein increased in the intermediary or later phase of acute inflammation.

Conditions for purification of the ninth component of bovine complement (C9) were established. The conditions for binding and elution from diethylaminoethyl cellulose and hydroxylapatite were different than for human C9. Serum albumin, a frequent contaminant of bovine C9 preparations, was removed by chromatography on reactive-red agarose. The calculated molecular weight of bovine C9 was 66,000, and reduction with 2-mercaptoethanol affected its migration on polyacrylamide gel electrophoresis. Some preparations of bovine C9 migrated as 2 bands when partially reduced, but extensively reduced preparations had a single band.

During initial studies, we found that many fluorescein isothiocyanate-labeled anti-immunoglobulin conjugates were unstable and tended to aggregate and precipitate when used for indirect immunofluorescence microscopy. In some instances, the precipitate was extensive enough to interfere with interpretation of the test results. Attempts to resolve this problem resulted in a procedure by which such conjugates were digested with papain to Fab and Fc fragments before use. Aggregation and precipitation were prevented, while desired antibody activity was retained. Digestion with papain also reduced the diffuse background fluorescence (commonly referred to as nonspecific fluorescence or staining) that is often associated with conjugates before they are sorbed with tissue powders or chromatographed to remove highly labeled immunoglobulin molecules.