Quantitative determination of antibody against hepatitis B surface antigen: measurement of its binding capacity.
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Biomedical subjects
Publications and source records attributed to R Thomssen.
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IgM antibody to hepatitis B core antigen (anti-HBc) was determined by a reverse enzyme immunoassay. In all of 58 patients with transient hepatitis B surface antigen (HBsAg)-positive acute hepatitis, IgM anti-HBc was detected in high titer. In 79.3%, IgM anti-HBc disappeared within two years, but in the remaining 12, it was still detectable. In 20 of 21 patients who developed chronic hepatitis, IgM anti-HBc was present after two years. High titers of IgM anti-HBc were found in 13 patients with histologically confirmed hepatitis B in whom HBsAg could not be detected in the initial serum samples; 11 of them later developed antibody to HBsAg and/or e antigen. Furthermore, IgM anti-HGc was detected in eight (5.6%) of 142 HBsAg-negative blood donors with elevated levels of serum transaminases and in 11 (0.5%) of 2,400 HBsAg-negative blood donors with normal levels of serum transaminases. Thus, IgM anti-HBc might be a better indicator of hepatitis B than HBsAg and help differentiage acute from chronic infection in HBsAg-positive patients.
Hepatitis B surface antigen (HBsAg) was purified from human plasma by gel chromatography, isopyknic centrifugation, and zonal centrifugation. The final product had about 60% of the original activity and was essentially free from hepatitis B virus particles (HBV) and plasma proteins. Treatment with formaldehyde concentrations up to 0.1% for inactivation of residual infectivity did not significantly reduce antigenicity in vitro and immunogenicity in guinea pigs. Adsorption to aluminum hydroxide resulted in 16-fold higher concentrations of antibody against HBsAg (anti-HBs) than did injection of soluble HBsAg. After two injections of 0.2 microgram HBsAg, which was treated with 0.1% formaldehyde and absorbed to aluminum hydroxide, the median titer of anti-HBs in guinea pigs was 4 IU/ml (normal value in human hepatitis B convalescents: about 0.1) for 1 year without further injections. When guinea pigs received 12 equivalents of homologous anti-HBs serum before the first injection of adsorbed HBsAg, the same anti-HBs titers were found after the booster injection as in animals which had not been passively immunized. A simultaneous application of an experimental HBsAg vaccine and hepatitis B immunoglobulin would probably decrease the potential risk of HBV infections caused by the vaccine itself and also produce rapid protection. To establish absence of HBV as completely as possible, the vaccine should be produced from anti-HBe-positive plasma by efficient purification procedures and it should be inactivated by formalin.
The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA. The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa, I, and Xho and separation of fragments on agarose gels. The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both. Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains. cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.
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The development of cytopathogenic changes in chicken embryo fibroblasts infected with the herpesvirus of the turkey, strain PB-THVI, and the release of virus particles into the supernatant of infected cultures is accelerated at temperatures higher than 37 degrees C and is fastest at 41 degrees C, the normal body temperature of chickens. The growth rate of HVT in CEF cultures was followed by determination of the number of virus genome equivalents within infected cells at various time intervals p.i. A temperature-dependent increase in the amount of virus DNA per infected cell could be detected, which is highest at 41 degrees C. At all temperatures tested (34, 36, 41 and 43 degrees C) the number of virus genome equivalents per infected cell ultimately reaches the same level. In the course of infection, virus DNA in CEF cultures at 37 and 41 degrees C becomes associated with the cellular DNA, as determined by neutral CsCl gradients of the total cellular DNA and hybridization of each fraction with 32P-labelled virus-specific complementary RNA. Association of virus to cellular DNA occurs earlier at 41 than at 37 degrees C. However, the same proportion (45%) of the total virus DNA appears ultimately to be associated with cellular DNA at both temperatures. A temperature-shift of CEF cultures infected with PB-THVI from 41 to 37 degrees C 24 h p.i. resulted in the same replication kinetics of virus DNA as was found at 41 degrees C.
The frequency of hepatitis B was examined using three serological parameters: HBsAg, antiHBs and antiHBc. All three substances were detected qualitatively by means of sensitive radioimmunological techniques. Of 1216 patients with acute hepatitis, 55 percent were HBsAg and antiHBc positive on admission to hospital. A further 17.8 percent had no HBsAg but were antiHBc positive and also partly antiHBs positive. These cases can be divided into 2 groups: In one group, in 8.6 percent of patients had a high antiHBc concentration during the acute phase. Similar antiHBc concentrations were seldom found (0.04 percent) in HBsAg negative blood donors. AntiHBs in the patients was at first mainly negative and then appeared during reconvalescence. These cases were considered to be acute hepatitis type B, although HBsAg was absent. In the second group, comprising 9.2 percent of the patients, antiHBc was present in low concentrations and in majority of cases antiHBs had been present from the beginning. The same antibody constellation was found in 3.6 percent of 2341 blood donors. In the group of patients it is supposed that the acute hepatitis present is not of type B and has a different aetiology. The low concentration of antibody is interpreted to be a sign of an earlier HBV-infection.
A specific and sensitive solid-phase radioimmunoassay has been developed for the detection of LCM virus antigens and anti-LCM virus antibodies. The test was performed in a microtiter system using polyvinylcholoride wells coated with anti-LCM virus rabbit hyperimmune serum. LCM virus antigens were allowed to bind to this antibody and afterwards detected by 125J-labeled anti-LCM virus-gamma-globulin. Anti-LCM virus antibodies were assayed by specific inhibition of these bound antigens. Since this technique is rapid and easy to perform the solid-phase radioimmunoassay is a valuable test for detecting LCM virus infections. Selected at random, 208 girls with, and 208 girls without, contact to Syrian hamsters were investigated for anti-LCM virus antibodies. Five (2.4%) of the hamster-exposed girls had anti-LCM virus antibodies (RIA-titer 1:8-1:32) in contrast to none of the control group (P=0.03).
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Specific recognition of antigens by cytolytic T lymphocytes sensitized to vaccinia virus was tested by monolayer adsorption. Adsorption was possible only on monolayers also expressing syngenic H-2 as viral antigens present during the sensitization phase. Vaccinia-virus-infected target cells were subjected to papain and neuraminidase treatment. H-2 antigenic determinants could be removed by papain treatment. Due to virus-specific inhibition of host-cell protein synthesis, reexpression of H-2 antigenic determinants did not take place, but viral surface antigens were resynthesized. Susceptibility of target cells to T-cell-mediated lysis was decreased after papain treatment. Substrains of vaccinia virus were used in order to define the minimal changes induced by vaccinia virus necessary for T-cell sensitization in vivo and target-cell lysis in vitro. When the immune response to a conditioanl lethal mutant strain of vaccinia virus was investigated, it could be demonstrated that expression of early surface antigens is sufficient for induction of the cellular immune reactions. These data were confirmed by inhibition studies with virus-specific antisera.
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Peroxidase-conjugated anti-human immunoglobulin was used to determine the percentage of B lymphocytes in the mononuclear cell fraction of peripheral blood of 100 healthy persons (blood donors). Peroxidase activity was revealed by incubation with the usual mixture of 3'3 diaminobenzidine and hydrogen peroxide. Cells which were peroxidase-positive after incubation with benzidine solution alone were considered to be monocytes. The number of T lymphocytes was estimated by the formation of E rosettes. 19.6% mononuclear cells could be shown to be B cells, 4.1% were monocytes and 57.0% T cells. The use of peroxidase-conjugated anti-human immunoglobulin gives rise to the same percentage of B cells as the use of fluorescein-conjugated anti-human immunoglobulin. The advantage of the method described here is that B lymphocytes can be counted by conventional light microscopy.
LCM virus, strain WE--grown on L cells--and labeled with 3H-uridine was centrifuged to equilibrium in a sucrose density gradient and examined in fractions for infectivity, incorporated radioactivity, and electron-microscopic features. The peak of infectivity is congruent with the one of radioactivity (density = 1.17 g/ml). LCM virus specificity of the radioactive peak was proved by precipitation of the radioactivity with anti-LCM virus antiserum. The peak fractions showed an abundance of 106 +/- 14 nm (1s) particles. They could be agglutinated with specific anti-LCM virus antiserum but not with antiserum directed against the histocompatibility (H-2) antigens of L cells.
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Many investigators in Europe and the USA have usually found high rates of serum conversion after vaccination with the rubella vaccines Cendehill and RA 27/3, but the resistance of vaccinated individuals against superinfection without disease induced by wild virus strains under experimental or natural conditions seems to be very low; e.g. up to 80% of the vaccinees reacted with increasing titers of hemagglutination inhibiting antibodies after super-infection with a wild virus in a special epidemiological situation. In our two studies performed under natural conditions of infection including an observation period of three years resp. five years after vaccination a more favourable picture evolved, perhaps reflecting a different epidemiological situation, a different susceptibility or a less virulence of wild virus in our region: Three years after vaccination of 14 years old girls with Cendehill and RA 27/3 in a double blind trial a significant increase in the titers of hemagglutination inhibiting antibodies was observed after vaccination with Cendehill by the subcutaneous route in only 1,16% (1/86) and in 1,18% (1/85) after vaccination with RA 27/3 by the same route. In the same observation period the infection rate in a control group not protected by natural immunity or by vaccination was 54,72% (87/159) and in a second control group, protected by natural immunity 1,82% (3/165)...
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