Functional and molecular characterization of a monoclonal antibody against the 165-186 peptide of human IL-1 beta.

A synthetic peptide of human recombinant interleukin 1 beta (hrIL-1 beta) 165-186, which exhibits biological activity in the human fibroblast assay, was used as an immunizing antigen to obtain a murine monoclonal antibody (MoAb) termed FIB 1. This MoAb, an IgG1, reacts specifically with hrIL-1 beta, but not with hrIL-1 alpha, as measured in enzyme-linked immunosorbent assays (ELISA). The MoAb FIB 1 detects the characteristic 17 kDa IL-1 protein in Western blots. Binding to the antigen is specific, as deduced also from the close correlation of ELISA immunoreactivity with IL-1 biological activity. The anti-IL-1 beta 165-186 Ab specifically neutralizes the biological activity of hrIL-1 beta and native IL-1, as measured by the IL-1-induced proliferation of murine thymocytes and human fibroblasts and the IL-1-dependent IL-2 production by murine T cells (EL4-6.1). Fifty per cent of hrIL-1 beta activity (25 U/ml, or 0.25 ng/ml) has neutralized by less than 30 micrograms/ml of MoAb. Furthermore, FIB 1 recognizes intracellular IL-1 in lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The anti-IL-1 beta 165-186 Ab does not react with the shorter IL-1 beta fragment 161-173 in solid-phase ELISA, therefore the binding region seems to be localized in the amino acid sequence VALGLKEKNLYLS. A sandwich-ELISA, using a polyclonal sheep anti-IL-1 beta 251-269 Ab as the capture antibody and an anti-IL-1 beta 165-186 MoAb as the detecting probe, allowed the determination of IL-1 beta from crude culture supernatants.

Identification and behavior of the precipitating BLV antibodies in sera of leukotic cattle.

Precipitating bovine leukemia virus antibodies isolated from bovine leukemic sera by ion-exchange chromatography, gel filtration and affinity chromatography were identified as immunoglobulins of the IgG1 subclass and IgA class. They proved to be different with regard to molecular size and electrical charge. Immunoglobulins IgG2 and IgM as well as low-molecular components ranging in the microglobulin regions (less than 4S) failed to precipitate BLV antigens. Individual bovine leukemic sera showed differing precipitating activities against p24 and gp69 antigens. Precipitating monospecific gp69 antibodies represented exclusively the IgG1 subclass. Some unidentified antigens in disrupted BLV preparations showed serological relationship to p24 and gp69, respectively.

Subunit and fine structure of the glycoprotein of bovine leukemia virus.

The structure of the glycoprotein of bovine leukemia virus (BLV) was investigated. Analysis of BLV under nonreducing conditions revealed two viral glycoproteins with apparent molecular weights of 162,000 (VGP I) and 85,000 (VGP II). After reduction and alkylation, two glycosylated polypeptide chains of apparent molecular weights of 60,000 (gp 60) and 30,000 (gp 30) could be demonstrated in VGP I and VGP II; VGP I appears to contain two molecules of gp 60 and two molecules of gp 30, and VGP II consists of only one molecule of gp 60 and one molecule of gp 30 linked by disulfide bonds. Comparison of the tryptic peptides of gp 60 and gp 30 provided evidence for the existence of partial sequence relationship between gp 60 and gp 30. The structural similarity of the BLV glycoprotein molecule to immunoglobulins is discussed in regard to the possible function of this protein in the pathogenesis of bovine leukemia.

Sequential studies of bovine leukemia virus antibody development in dairy cattle over a four-year period.

Serological examinations using the agar gel immunodiffusion and immunofluorescence tests were undertaken to study: a) the development of bovine leukemia virus infections in a given herd under field conditions monitored by antibody development, b) the reproducibility of the results of serological examination during 22 sequential tests, c) the antibody titre variations amongst individual animals. A total of 286 Friesian cattle representing 2,352 serum samples was tested at regular intervals over a period from 1974/75 to 1978. The percentage of animals with bovine leukemia virus antibodies continuously increased during the observation period from an initial 4.8% (1974/75), 9.7% (1976), 34.2% (1977) to 52.6% (1978). On the basis of 22 sequential tests representing 1,672 serum samples obtained from 76 animals no false positive reactions were seen in the immunodiffusion test. Thirty-six (14.2%) out of 254 animals, however, showed variations of the antibody titre leading to a shift from positive to negative or negative to positive results, respectively. AnothFr three cattle (1.2%) shifted from a positive to negative immunodiffusion result and maintained their negative reactivity for at least two following tests. But they still had specific antibodies if tested by the immunofluorescence test. A detailed analysis of the group of variable reactors supports the view that a more sensitive and quantitative test system may be helpful to complement the presently used immunodiffusion test for the eradication of enzootic bovine leukosis.

Purification of a glycoprotein from bovine leukemia virus (BLV).

A precipitating antigen of bovine leukemia virus was isolated by isoelectric focusing and Sephadex gel filtration. In SDS-polyacrylamide gel electrophoresis it was found to be a homogeneous protein with a relative molecular weight of 69000 daltons. Because of its relative molecular weight and staining characteristics it was designated as BLV gp69. A protein with the same molecular weight could also be demonstrated in BLV particles. In 34 out of 35 sera from cattle affected by enzootic bovine leukosis antibodies against gp69 were detected, whereas the sera from 197 animals, free of bovine leukosis, did not react in immunodiffusion test.

The conservation of poly-A-containing RNA during the dormant state of the moss Polytrichum commune.

The moss Polytrichum commune can be dried to less than 2% of free water and kept for some weeks without losing its viability. Upon rehydration of the moss, protein synthesis starts about 60 minutes before incorporation of radioactive precursors into RNA can be observed. Pulse labeled total RNA and poly-A-containing RNA do not show quantitative or qualitative alterations during desiccation up to 20 days.