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Biomedical subjects

G Jahn

Publications and source records attributed to G Jahn.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cleavage and purification of prokaryotically expressed HIV gag and env fusion proteins for detection of HIV antibodies in the ELISA.

Parts of the gag p24 and the gp41 transmembrane protein of the human immunodeficiency virus HIV-1 were expressed as fusion proteins in Escherichia coli, using an expression vector carrying aa 1-375 of the lac-Z gene linked to the recognition sequence for the blood coagulation factor Xa. Fusion proteins were cleaved into the bacterial and viral portion and the viral polypeptide was purified by a molecular sieve column. The purified viral antigens were tested with 288 human sera in the enzyme-linked immunosorbent assay (ELISA) technique. Comparison with commercially available tests showed comparable sensitivity and a higher specificity of the gag/env-ELISA for borderline reactive sera.

Chromatography, Gel↗
PubMed1991

Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method.

Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

Adult↗
PubMed1990

Generation and application of a monoclonal antibody raised against a recombinant cytomegalovirus-specific polypeptide.

Procedures for diagnostics of cytomegalovirus infections include histopathology, cell culture, serology, and direct detection of viral antigens or nucleic acids within infected cells or tissues. In order to develop a new diagnostic reagent for viral antigen detection, we generated a mouse monoclonal antibody. This antibody was raised against a recombinant antigen representing part of the large phosphorylated structural protein pp150 of human cytomegalovirus. The monoclonal antibody was shown to be useful for antigen detection by immunofluorescence and immunoenzymatic staining in infected cells from cell culture as well as from infected organs. The antibody proved to be reactive even in paraffin-embedded sections from tissue specimens.

Antibodies, Monoclonal↗
PubMed1990

Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1.

The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as beta-galactosidase fusion proteins, covering about 75% of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.

Antibodies, Monoclonal↗
PubMed1990

Procaryotic expression of phosphorylated tegument protein pp65 of human cytomegalovirus and application of recombinant peptides for immunoblot analyses.

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons, termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immune response during natural infection. A monoclonal antibody directed against this protein was used to screen a lambda gt11 cDNA library for recombinant polypeptides. Two DNA fragments from purified lambda clones and one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. The resulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed against pp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodies tested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids. Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen for serodiagnosis but may be very useful in combination with other HCMV proteins.

Animals↗
PubMed1990

Large-scale screening of human sera with cytomegalovirus recombinant antigens.

One of the major problems regarding cytomegalovirus (CMV) serodiagnosis is the use of poorly defined viral antigens. Individual CMV proteins expressed via recombinant DNA procedures are a promising approach to solving this problem. In this work, 10 different fusion proteins containing antigenic epitopes of the major CMV structural proteins of 150, 71, 65, 38, and 28 kilodaltons and of the nonstructural protein of 52 kilodaltons were subjected to Western (immuno-) blotting to assay their reactivities with immunoglobulins G and M in 395 CMV-seropositive and 100 CMV-seronegative unselected human serum samples. As a whole, the results obtained indicate that CMV can be replaced by recombinant viral proteins in the serological evaluation of anti-CMV antibodies.

Antigens, Viral↗
PubMed1990

Transcriptional analysis of the eight-kilobase mRNA encoding the major capsid protein of human cytomegalovirus.

The 8-kilobase mRNA coding for the major capsid protein (MCP) of human cytomegalovirus was precisely mapped. Two 5' ends of the transcript were located within HindIII fragment a, 29 and 34 base pairs, respectively, downstream of the sequence TATTAGA. The 3' end was localized within HindIII fragment b of the viral genome. The MCP transcript was synthesized at late times after infection and was not detected before viral DNA replication. In addition, the MCP promoter region could be identified, which strongly responded to viral trans activation at early and late times after infection in a transient expression assay.

Base Sequence↗
PubMed1989

Discrimination of HIV-1 and HIV-2 infection based on recombinant immunoblots.

Recombinant polypeptides representing various parts of structural proteins of HIV-1 and HIV-2 were expressed in E. coli. Fragments of the transmembrane proteins gp41 of HIV-1 (HTLV-IIIB) and gp38 of HIV-2 (LAV-2 ROD) proved to be highly antigenic in the immunoblot test system. Each protein was found to be suitable for detection and differentiation of antibodies in sera of patients infected with HIV-1 or HIV-2. Sera of 100 patients from West-Germany, which were confirmed as positive for HIV-1 antibodies, reacted clearly with the HIV-1 recombinant antigen (fpOE-6); no or only weak immune reactions were seen when the analogous recombinant HIV-2 antigen (fpOE-T) was used in the same immunoblot test. An inverse reaction pattern was found with 7 sera from Africa which, by conventional means, were proved to be HIV-2 antibody positive. These sera specifically reacted with the homologous HIV-2 fusion protein and no or only weakly stained bands were identified on the analogous strip with the HIV-1 antigen (fpOE-6).

Adult↗
PubMed1989

Hybridization techniques provide improved sensitivity for HCMV detection and allow quantitation of the virus in clinical samples.

Hybridization techniques (slot-blot and in-situ hybridization assays) and immunostaining using murine monoclonal antibodies directed against different proteins of the human cytomegalovirus (HCMV) were compared for their sensitivity and specificity for detection of HCMV. A model system with HCMV infected human embryonic lung fibroblasts and lung biopsy specimens obtained from patients with culture positive HCMV interstitial pneumonia were used for evaluation of these techniques. The hybridization techniques were found to provide an improved sensitivity compared to immunostaining. Additionally a good correlation was found between the virus dose determined by TCID50 and the amount of viral DNA detected by slot-blot hybridization and by the number of autoradiographic silver grains per 100 cells per 2 weeks exposure time detected in the infected fibroblasts by in-situ hybridization. Thus, at least in the model system quantification of the virus was achieved by hybridization assays.

Cells, Cultured↗
PubMed1989

Multiplicity dependent expression of the predominant phosphoprotein pp65 of human cytomegalovirus.

Recent clinical isolates of human cytomegalovirus (HCMV) display considerable quantitative differences in the expression of the structural phosphoprotein pp65. This study shows that a reduced production of pp65 correlates with reduced amounts of its coding transcript in wild-type strains compared to the high level expression in the laboratory strain AD169. The cleavage pattern of the encoding DNAs including the promoter region did not show major differences among the isolates. Therefore the variable expression of pp65 is supposed to be dependent on cell culture conditions. Increasing multiplicities of infection resulted in overproduction of pp65 and this may lead to the increased formation of dense body particles.

Blotting, Northern↗
PubMed1989

Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays.

The proteins p15 and p24 of the human immunodeficiency virus (HIV) type 1 gag gene were expressed as fusion proteins in Escherichia coli for detecting antibodies against the acquired immunodeficiency virus by Western blot (immunoblot) analysis. These fusion proteins contain amino acids 1 to 375 of the E. coli beta-galactosidase linked to the viral protein(s) by a recognition sequence for the specific protease factor Xa. They are obtained in large amounts in insoluble inclusion bodies. To avoid ambiguous results caused by cross-reaction of sera with bacterial proteins in Western blots, we purified the recombinant fusion proteins and subsequently removed the bacterial part of the fusions by cleavage with factor Xa. The cleavage mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The viral proteins obtained by this method did not contain any bacterial proteins or protein fragments. Thus, false-positive results in HIV Western blot analysis with bacterially expressed HIV proteins can be excluded with these purified recombinant viral antigens.

Amino Acid Sequence↗
PubMed1989

Identification of the major capsid protein gene of human cytomegalovirus.

The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus. The predicted length of the HCMV MCP was 1,370 amino acids. Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus. A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene. Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases. A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits. The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions. A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots.

Amino Acid Sequence↗