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.

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.

: Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method. Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence. To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems. Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced in E. coli as stable immunogenic beta-galactosidase fusion proteins. Recombinant proteins were taken for immunoblot-assays. The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins. Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins. Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.

Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method. Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence. To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems. Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced inE. coli as stable immunogenic β-galactosidase fusion proteins. Recombinant proteins were taken for immunoblot-assays. The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins. Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins. Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.

Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method. Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence. To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems. Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced in E. coli as stable immunogenic beta-galactosidase fusion proteins. Recombinant proteins were taken for immunoblot-assays. The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins. Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins. Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.