Detection of yellow fever virus in serum by enzyme immunoassay.

Yellow fever (YF) virus is present in patient's blood during the acute phase of illness. Virus isolation and identification provide a potential method of early diagnosis, but available techniques are slow and require specialized materials and equipment. An alternative approach is direct detection of YF antigen in serum by means of an enzyme-linked immunosorbent assay (ELISA). An antigen-capture ELISA was developed, which used anti-YF antibodies, immobilized on a solid phase (polystyrene plates), to capture YF virus from serum samples. After addition of the virus-containing sample, anti-YF detecting antibody conjugated to alkaline phosphatase was added to detect viral antigen. Trials with various capture and detecting antibodies in systems employing purified YF 17D virus, led to the selection of: 1) two capture antibodies (pooled human serum containing high titer YF IgM antibodies and a type-specific YF monoclonal antibody), and 2) a detecting antibody conjugate consisting of monoclonal antibody broadly cross-reactive with all flaviviruses, purified by affinity chromatography, and conjugated to alkaline phosphatase. The limit of sensitivity in tests against purified YF 17D virus diluted in buffer or normal human serum was 10(3.0) - 10(3.6) PFU/0.05 ml or 0.007-0.029 microgram viral protein/0.05 ml. Sera obtained at intervals from rhesus and cynomolgus monkeys after infection with a wild YF virus strain were tested. The limit of sensitivity of the assay applied to viremic monkey serum was similar (approximately 3.5 log10PFU/0.05 ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytological changes occurring in the liver of coturnix quail with an acute arsenic exposure.

Coturnix quail (Coturnix coturnix) were given acute oral doses of sodium arsenite (NaAsO2). On each of two successive days they received 1 mg sodium arsenite, followed by 3 mg on the third day. There was no observable difference between the arsenic-exposed quail liver and the controls at the light microscope level; however, swelling of granular endoplasmic reticulum (GER) in the hepatocytes of the arsenic-exposed quail was detected with electron microscopy. The swollen organelles were determined to be GER by their peripheral orientation around the mitochondria and the ribosomes attached to their cytoplasm-exposed membrane surfaces. The degree of swelling ranged from slight to severe, and was due to an osmotic imbalance possibly brought about by the indirect inhibition of the sodium pump by arsenic.

Disruption of active zones in frog neuromuscular junctions following treatment with proteolytic enzymes.

Frog neuromuscular junction treated with proteolytic enzymes to remove the basal lamina were studied with freeze-fracture techniques in order to examine the influence of the basal lamina in the maintenance of active zone ultrastructure. The active zone is believed to be the site of transmitter release and has a unique membrane organization and location in the neuromuscular junction. After removal of the basal lamina by successive treatment of 0.01% collagenase and 0.1% protease for 1 h each, active zone disruption was observed. Some active zones became segmented, and some were also randomly located and oriented, but they still had normal double-row particle organization. Others contained only clusters of large intramembrane particles. These disorganized active zones were still functional as indicated by the presence of vesicle openings. Some enzyme-treated junctions were also exposed to the membrane cholesterol probe, filipin, to examine the expression of membrane lipid heterogeneity in disrupted active zones. As in normal active zones, filipin-sterol complexes were absent. The densities of background particles in the presynaptic membranes and of large particles thought to be acetylcholine receptors were not significantly altered by the enzyme treatment. Although a direct effect of the enzymes on active zone ultrastructure can not be totally excluded, the present work is consistent with a maintenance role of the basal lamina in active zone organization and location.

Immunoglobulin M antibody capture enzyme-linked immunosorbent assay for diagnosis of St. Louis encephalitis.

Sera from patients with St. Louis encephalitis were tested with an immunoglobulin M (IgM) antibody capture enzyme immunoassay (MAC ELISA). The assay used five reagents: antihuman IgM, test serum, sucrose-acetone-extracted mouse brain antigen, broadly cross-reactive flavivirus monoclonal antibody conjugated to alkaline phosphatase, and substrate (p-nitrophenyl phosphate). MAC ELISA endpoint titers correlated (r = 0.893) with the absorbance value of a 1:100 dilution of patient serum. Significant (fourfold or greater) changes in the endpoint titers between paired sera corresponded to a critical ratio (ratio of absorbance values at the 1:100 dilution) of greater than or equal to 1.3. IgM antibodies were detected in 71% of patients bled at 0 to 3 days after the onset of illness, in 99% bled at 4 to 21 days, and in 91% bled at 22 to 67 days. Thereafter, the IgM seropositivity rate declined; however, 29% of sera were still positive at 115 to 251 days after the onset of illness. MAC ELISA titers were significantly correlated with hemagglutination inhibition (r = 0.258) and neutralization (r = 0.711) titers. Because IgM antibodies appeared early and waned rapidly, a diagnosis was made on the basis of a decrease in titer more often by MAC ELISA than by hemagglutination inhibition, complement fixation, or neutralization tests. IgM antibodies generally showed a high degree of specificity; heterologous cross-reactions were, however, present in 4 of 14 sera examined. The MAC ELISA is useful for the rapid, early diagnosis of St. Louis encephalitis.