Biochemical and immunological characterization of structural proteins from retrovirus-D/New England and comparison to Mason-Pfizer monkey virus and permanent human fibroblast virus.

Biochemical and immunological properties of retrovirus-D/New England (here referred as R-D/NE) recently isolated at the New England Regional Primate Research Center, Southborough, Ma, from a rhesus monkey with acquired immune deficiency syndrome were investigated and compared to the prototype type D retroviruses Mason-Pfizer monkey virus (MPMV) and permanent human fibroblast virus (PMFV) isolated from a breast carcinoma of a rhesus monkey and a continuous human cell line, respectively. The polypeptide composition of R-D/NE propagated in a human lymphoid B cell line (Raji cells) has been investigated using SDS-polyacrylamide gel electrophoresis. Staining with Coomassie blue and labelling with 14C amino acids revealed seven viral polypeptides with molecular weights of 4,000, 10,000, 12,000, 15,000, 18,000, 27,000, and 80,000 Da which were also shared by MPMV and PMFV. The 80,000 Da protein was shown to be a glycoprotein by incorporation of 3H glucosamine. The 18,000 Da protein was identified as a phosphoprotein of R-D/NE. p18 structural proteins of MPMV and PMFV represent phosphoproteins of their respective viruses as well. All three phosphorylated proteins contain O-phosphoserine as major phosphoamino acid. The comparison of tryptic peptide maps of the major internal structural proteins of R-D/NE, MPMV, and PMFV revealed a striking similarity among p 10/p 12 and p 15. proteins. A minor difference was detected among the tryptic peptide digests of p 4 and p 27 proteins. Antiserum against p 15 of MPMV showed a significantly weaker binding to R-D/NE than to MPMV and PMFV at high dilutions.

Effect of islet cell surface antibodies on neonatal rat pancreatic islet cells or isolated islets of Langerhans in vitro.

Islet cell surface antibodies (ICSA) raised by immunizing rabbits with pancreatic islet cell suspensions were characterized with respect to some aspects of their influence on neonatal rat islet cells or isolated islets of Langerhans. The removal of unspecific cytotoxic factors by absorption with liver powder and spleen cells was reflected in changes of the antibody binding pattern to islet cells demonstrated by flow-cytometric analysis as well as the corresponding 51Cr-release from prelabelled islet cells. Using neonatal rat pancreatic islets as a target, fresh ICSA-positive serum provokes a beta-cell specific insulin leakage in a concentration dependent manner. In contrast to heat-inactivated antiserum the complement-mediated cytotoxic effect of rabbit anti-rat islet cell surface antiserum seems to have its morphological expression also in alterations of the islet surface structure as revealed by scanning electron microscopy.

[Characterization of monoclonal antibodies to human monocytes]. / Charakterisierung von monoklonalen Antikörpern gegen humane Monozyten.

A set of monoclonal antibodies reacting with human peripheral blood monocytes is characterized. Two of these antibodies also bind to a fraction of granulocytes (BL-M/G-1) respectively to all of them (BL-M/G-2). On the other hand the BL-M-3 appears to be specific for monocytes only. All three monoclonals do not react with enriched B and T lymphocytes, platelets, erythrocytes and several lymphoid or erythroid permanent cell lines. The antigens recognized by all three monoclonals are expressed by monoblastic cell line U-937, whereas the myeloid line HL-60 and the cell line KG-1 express only antigen recognized by monoclonal antibody BL-M/G-2.

The detection of distinct epitopes of human alpha-fetoprotein (AFP) by solid phase radioimmunoassay using hybridoma culture fluid.

A solid phase radioimmunoassay was developed for the epitope analysis of human alpha-fetoprotein (AFP) using hybridoma culture fluids. The following incubation sequence was performed: anti-mouse Ig, hybridoma culture fluid, normal mouse serum, and a mixture of 125I-labeled AFP and hybridoma culture fluid. Seven epitopes were detected by monoclonal antibodies produced by 15 presumably independent hybridoma clones.

Comparison of monoclonal and polyclonal antibodies in a two-site binding enzyme immunoassay for alphafetoprotein (AFP).

A two-site binding enzyme immunoassay for the detection of alphafetoprotein (AFP) was developed by using either a combination of two monoclonal antibodies or of one monoclonal antibody and polyclonal antibodies. The conjugation of the monoclonal antibodies to peroxidase by the periodate method yielded a somewhat higher sensitivity in the enzyme immunoassay (EIA), when compared to conjugates produced by the glutaraldehyde method. The detection limit was 10 micrograms AFP per litre when using only monoclonal antibodies in the assay. Simultaneous incubation of the sample and the monoclonal labelled antibody should be avoided unless using at least two different dilutions of the sample for investigation.

Monoclonal antibodies to different epitopes of human alpha-fetoprotein (AFP).

Monoclonal antibodies specific for human alpha-fetoprotein (AFP) were produced by the hybridoma technique. By using solid-phase immunofluorescence and radioimmunoassays, the antibodies were proven to distinguish 4 different epitopes on the AFP molecule.

Determination of immunoglobulin class and subclass of monoclonal antibodies to human alpha-fetoprotein by solid phase radioimmunoassay.

A solid phase radioimmunoassay using the incubation sequence: mouse immunoglobulin (Ig) rabbit anti-mouse Ig, monoclonal antibody and 125I-labeled antigen was performed to determine the class and subclass of seven murine monoclonal antibodies against human alpha-fetoprotein. All antibodies belonged to the IgG class. The subclass type was IgG 2a for four antibodies, IgG 1 for two antibodies and IgG 2b for one antibody.

A solid-phase immunofluorescence assay (SIFA) using membrane filters.

A solid-phase immunofluorescence assay (SIFA) is presented which allows the demonstration of antibodies against soluble antigens. The test is performed by successively incubating nitrate cellulose discs in microtitration plates with anti-Ig, the sample to be tested and FITC-labeled antigen. The fluorescence intensity of the discs is then measured by using a microfluorometer. The assay is simple, needs a small amount of material and allows to test a large number of samples.

Use of Sepharose bead immunofluorescence assay for comparison of the type D retroviruses MPMV and PMFV.

Mason-Pfizer monkey virus (MPMV) and PMFV, an isolate from a human continuous cell line, were compared by Sepharose bead immunofluorescence assay. According to the results with p27-specific assays the main structural protein of both viruses seems to be identical in the prominent antigenic determinants. Differences were found when comparing the p15s indicating that this viral protein contains type-specific antigenic determinants.

Monoclonal antibodies against different antigenic determinants of a protein molecule (human serum albumin) as detected after labeling the antigen with two different markers.

By using FITC- and 125I-labeled human serum albumin (HSA) the reactivity of anti-HSA hybridoma antibodies was compared in solid-phase immunofluorescence assay (SIFA) and radioimmunoassay (SRIA). A considerable number of hybridoma antibodies had been detected which are positive in SIFA but not in SRIA. Since this cannot be explained by a difference in the sensitivity of the assays it seems likely that these monoclonal antibodies detect antigenic determinants which contain tyrosine as an essential part.

The application of a sepharose bead immunofluorescence assay and a solid-phase radioimmunoassay to the bovine leukemia virus system.

Several fluorescence assays with bovine leukemia virus (BLV) conjugated to activated Sepharose 4B were used for the detection of BLV and anti-BLV antibodies. These tests were compared with a solid-phase radioimmunoassay and found to be in the same sensitivity range. Sepharose bead immunofluorescence assay and solid-phase radioimmunoassay can be applied to the diagnosis of BLV infection in cattle.

A solid-phase immunofluorescence assay (SIFA) for screening antigen-specific hybridomas.

A solid-phase immunofluorescence assay (SIFA) is described for screening of monoclonal hybridoma antibodies against soluble antigens. The test is simple, needs a small amount of material (20-100 ng of labeled antigen per sample) and allows a large number of samples (500-1 000) to be tested in 2 days. The assay is performed in microtitration plates with small cellulose nitrate discs as the solid phase. Anti-mouse IgG, the sample to be tested and FITC-labeled antigen are successively incubated with the discs. The fluorescence intensity of the surface of the discs is then measured with a microfluorometer. The assay has been used to select hybridomas against human serum albumin. Comparison of the immunofluorescence assay with a radioimmunoassay using conventional antisera and hybridoma antibodies similar sensitivity for both assays.

A microfluorometric immunoassay for the demonstration of human AFP.

A competitive Sepharose bead immunofluorescence assay is described which allows the quantitative detection of human alpha-fetoprotein. The test is highly specific and sensitive. The smallest demonstrable concentration of AFP is about 10 ng/ml.

Interspecies antigenic determinants of structural proteins of mammalian type-C viruses as detected by a competitive sepharose bead immunofluorescence assay.

The present paper describes a competitive immunoassay using antiserum to FeLV p 27 and SSV-conjugated Sepharose beads. The assay is applied to compare the interspecies-specific antigenic determinants of the major structural proteins of type-C viruses of different mammalian species. The test proves to be highly sensitive and specific and may be used for the demonstration of viral proteins in crude cellular extracts.