Biomedical subjects
Publications and source records attributed to W H Gerlich.
The minor glycoproteins from hepatitis B surface antigen, GP33 and GP36, contain at their carboxy-terminal part the sequence of the major protein P24. They have 55 additional amino acids at the amino-terminal part which are coded by the pre-S region of the viral DNA.
The protective effect of anti-HBs against hepatitis B virus is proven only for the common antibody anti-HBs/a but not for subtype specific antibody. Using subtype specific radioimmunoassays, anti-HBs/a and anti-HBs/d were quantitated in recipients of an HBsAg/ad vaccine. All persons developed anti-HBs/a. The relative proportion of anti-HBs/d was variable and very high at the beginning of the immune response. At this time the anti-HBs was predominantly in the IgM class. IgM-anti-HBs disappeared rapidly after its peak value and was more slowly replaced by IgG-anti-HBs. Persons who had only anti-HBs or anti-HBc as the only antibody did usually not react with an anamnestic booster response and developed IgM- anti-HBs after vaccination. An injection schedule of 0, 1, 4 months produced ten times higher titers than a 0, 1.5, 3 months schedule 4 weeks after the third injection. However, 6 months later titers were essentially identical. Nine of ten "non-responders" became positive after a fourth injection.
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No experimental setting is available to exclude residual infectivity in HBsAg vaccines derived from human plasma. Thus, safety can be achieved only by means of their preparation. To reduce infectivity of the starting material, only plasma from healthy anti-HBe positive donors was used. In the FRG, 50% of all healthy HBsAg carriers with anti-HBe have a suitable serum level of 5 to 20 micrograms/ml. The purification procedure removed hepatitis B virus by a factor greater than 10(4). The purified product contained only the HBsAg proteins and no serum protein, as shown by SDS gel electrophoresis. The pure HBsAg was treated with formalin 1:500 at 37 degrees C for 4 days. A loss of 30 to 50% antigenicity was tolerated to achieve the highest possible destruction of known and unknown infectious agents. After inactivation, the HBsAg was bound to aluminium hydroxide gel. The gel was washed repeatedly to remove the formalin. Doses of 40 micrograms or 20 micrograms absorbed HBsAg protein were given to greater than 2500 persons without serious side effects. In greater than 97% anti-HBs was formed with a median titer of 1900 I.U./ml.
The 20-nm particles of hepatitis B surface antigen (HBsAg) contain two minor glycoproteins, GP33 and GP36, which are probably encoded at their 55 N-terminal amino acids by the pre-s region of the viral DNA. Their 226 C-terminal amino acids are identical to the major protein P24. The 20-nm particles contained more GP33 and GP36 when the blood had a high HBsAg concentration. They were also found in relatively high amounts in HBsAg filaments and virions. Treatment with glycosidase and trypsin showed that the mannose rich glycan and the N-terminal portion of GP33 and GP36 were exposed at the surface of the HBsAg particles. The 20-nm particles containing much GP33 and GP36 did not induce higher anti-HBs titers in guinea pigs than those particles almost devoid of them.
Within one year four butchers fell ill with acute hepatitis B in a butcher's shop with ten employees. A symptom-free hepatitis B virus carrier was detected among the non-diseased employees. Lack of IgM-anti-HBc antibodies showed that the carrier state had probably existed for more than one year. The time course of infections and the kind of cooperation made it very likely that the carrier had infected his symptomatic mates. The outbreak shows that hepatitis B can be an occupational disease also in non-medical occupations. Should hepatitis B occur in a workman exposed to trauma frequently due to his occupation, serological investigation of the workmates is required in order to identify potential sources of infection and to enable future protective measures.
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The nature of the protein kinase (PK) which phosphorylates the core protein of hepatitis B virus in vitro was studied. The PK copurified with the core particles during rate zonal centrifugation and gel chromatography. It showed the same size heterogeneity as the core particles, which consisted of a main fraction of 28-nm particles and a subfraction of 22- to 26-nm particles. DNA-containing heavy core particles with a density of 1.33 to 1.35 g/ml and less endogenous PK than did the light cores. The phosphorylation reaction had a rapid initial phase (several minutes) and a slow but long-lasting second phase (many hours). The PK had a high affinity for ATP (KM = 0.5 mumol/liter). Only few of the several hundred P21.9 subunits in one core particle were phosphorylated in vitro. The only amino acid which was phosphorylated in vitro was serine. The resistance of the introduced phospho group against alkaline phosphatase showed that the PK acceptor, and probably the enzyme itself, was located inside the core particle.
Hepatitis B virus DNA contains a tightly bound protein which was not removed by healing to 60 degrees C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5' end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90 degrees C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5' end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5' ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5' end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.
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.
Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.
IgM antibody against core antigen of the hepatitis B virus (anti-HBc IgM) was selectively determined by a new enzyme immunoassay (EIA). Microtiter plates were coated with anti-human micro chain immunoglobulin. On addition of serum IgM is bound by a factor of about 4,000 more than IgG. After removing the sample, HBcAg is added to the IgM-coated surface. Binding takes place if the IgM contained anti-HBc and was demonstrated by the aid of a conjugate made from anti-HBc IgG and horse radish peroxidase. Quantitation may be achieved without testing a dilution series. The assay was not disturbed by a large excess of anti-HBc IgG in the sample and rheumatoid factor did not produce false-positive results, provided the sample was diluted in an excess of aggregated IgG. The diagnostic relevance of the assay was demonstrated in selected cases of acute hepatitis B. Rapid diagnosis of acute hepatitis B infection is therefore now possible in those cases whihc are HBsAg-negative but anti-HBc-positive.
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.
The large (pre-S1), middle (pre-S2) and major (P24) polypeptides of HBsAg have been defined in detail, but their role in hepatitis B virus infection is not known. Therefore, we studied the expression of pre-S1, pre-S2 and P24 in the liver of 15 patients with acute or chronic hepatitis B virus infection using monoclonal and polyclonal antibodies in a double staining immunofluorescence method. The pre-S and major HBsAg polypeptides were co-expressed in the hepatocyte cytoplasm of all patients except for one case of chronic active hepatitis and cirrhosis. HBcAg was present in hepatocyte nuclei of nine patients suggestive of active hepatitis B virus replication. These studies support the hypothesis that the pre-S polypeptides represent essential components of the envelope of hepatitis B virus.
The protein-blotting technique is a very practical way to detect and characterize certain proteins in a complex sample by various reagents like polyclonal and monoclonal antibodies, lectins, nucleic acids, or other ligands. This article discusses several options to perform this technique with special reference to viral hepatitis. We give a detailed description of denaturing gel electrophoresis, blotting and specific detection methods. At the beginning of each section a general outline of a certain technique is given, thereafter the reagents, equipment, suppliers, and techniques are described in a way that a beginner may be able to perform the assay.
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