Identification of a protein encoded in the EB-viral open reading frame BMRF2.

Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in the open reading frame BMRF2 we could identify a protein-complex of 53/55 kDa in chemically induced B95-8, P3HR1 and Raji cell lines. This protein could be shown to be membrane-associated, as predicted by previous computer analysis of the secondary structure and hydrophilicity pattern, and may be a member of EBV-induced membrane proteins in lytically infected cells.

New oligopeptide immunoglobulin G test for human parvovirus B19 antibodies.

A new, highly sensitive and specific enzyme immunoassay using oligopeptides as antigen (enzyme-linked immunosorbent assay [ELISA] B19-OP) for detecting parvovirus B19-specific immunoglobulin G (IgG) was established. As antigens, B19-specific oligopeptides of 24 and 30 kDa derived from a 196-kDa fusion protein of beta-galactosidase and viral capsid protein (VPI) of B19 after CNBr cleavage and separation by high-pressure liquid chromatography were used. Of 139 serum specimens tested in parallel for anti-B19 IgG by standard ELISA using B19 particles as antigen and by ELISA B19-OP, 73 (52.5%) were positive and 63 (45.3%) were negative in both tests, and 3 (2.2%) were negative by standard ELISA but positive by ELISA B19-OP and by immunoblot. By using ELISA B19-OP, it was possible to detect anti-B19 IgG in an asymptomatic blood donor 4 weeks after acute infection, and anti-B19 IgG titers of 10(-5) could be measured in convalescent-phase sera.

Purification of a recombinantly produced transmembrane protein (gp41) of HIV I.

The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies. The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli. The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell. Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures. Growth conditions of the recombinant E. coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen. Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components. For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used. After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed. The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important. Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.

Carrier-bound synthetic peptides. Use as antigen in HIV-1 ELISA tests and in antiserum production.

Chemically synthesize carrier-bound peptides have been used as antigens in diagnostic test systems (ELISA) and for raising antipeptide-specific antisera. The method does not require prior cleavage of the peptides from the support used for the solid-phase synthesis. Using the same resin for both the synthesis and the subsequent applications it was possible to avoid expensive and time-consuming purification procedures and artificial recoupling to solid supports. A quick and specific ELISA-based diagnostic test system for HIV-specific antipeptide antibodies in human sera was established. In addition the carrier-bound peptides were shown to be potent antigens for raising antibodies in animals.

Carrier bound synthetic oligopeptides in ELISA test systems for distinction between HIV-1 and HIV-2 infection.

A series of synthetic carrier bound oligopeptides derived from corresponding regions of the core and envelope proteins of HIV-1 and HIV-2 were used in enzyme-linked immunoabsorbent assays (ELISA) for serodiagnosis of HIV-1 and HIV-2 infected individuals. The combination of peptides from regions either conserved or highly variable between the two virus types allowed the identification of HIV infection in general and the differentiation between HIV-1 and HIV-2. No specific reaction was found in seronegative individuals. The use of peptides bound to the same polystyrene carrier as in peptide synthesis allowed the establishment of a highly specific and sensitive test system without the risk of unspecific cross-reaction due to contamination with bacterial or cellular protein material.

Use of synthetic oligopeptides in identification and characterization of immunological functions in the amino acid sequence of the envelope protein of HIV-1.

Following computer-assisted analysis of the amino acid sequence of various HIV-1 isolates, we synthesized a series of oligopeptides derived from variable and conserved regions of the envelope protein complex gp120/gp41. The peptides were used in ELISA tests for their reactivity with human antisera from HIV-1 positive individuals; patients with clinically manifested AIDS showed only a rather limited reaction, predominantly with two peptides (p102-112, p316-326), which is in contrast to sera from HIV-1 positive asymptomatic individuals, whose sera were reactive with almost all peptides. Using consecutive sera of the same patients, decreasing antibody titers to defined epitopes could be shown to occur during the development of AIDS. Cellular immune response recognition was analyzed in T-cell proliferation assays by [3H]thymidine incorporation. One peptide localized in a conserved region clearly induced proliferation of T-cells. Those data were combined to a map of the functions localized in the various regions of the HIV-1 envelope proteins.