Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

Radioimmunological analysis of the antigenic variability of influenza virus nucleoprotein

Influenza A virus ability to bind anti-NP monoclonal antibodies to two viral strains has been studied by radioimmunoassay on polyethylene film with the subsequent autoradiographic registration of results. Monoclonal antibodies were obtained to the viral strains differing in antigenic formula of outer glycoproteids and isolated at different time. The studied influenza viruses were divided into seven groups due to their ability to bind monoclonal antibodies. The absence of correlation between the antigenic properties of nucleoprotein and glycoproteids has been registered. Variability of some antigenic sites has been analyzed. The human epidemic strains of influenza virus are different in ability to bind monoclonal antibodies from the viral strains that are connected with animals in nature or laboratory practice.     

Probing eukaryotic RNA polymerases B with monoclonal antibodies

Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora comata were selected on the basis of different subunits recognition and inhibitory effect on enzyme activity. A library of 10 antibodies biased toward B180, B145, B39, B23,5 and B11 subunits was constructed. Most of these antibodies also recognize yeast, wheat germ and calf thymus RNA polymerase B. Subunits bearing antigenic determinants are not always homologous in Podospora and yeast enzyme. As some of these antibodies strongly inhibit enzyme activity they constitute potent probes for functional studies of corresponding subunits.     

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.     

Biological consequences of the decay of 3H and 14C incorporated into the influenza virus

An influenza virus labeled with 3H-uridine loses its infectiousness when stored for a long time. It is suggested that disintegration of tritium incorporated into virus RNA causes lethal intramolecular modifications therein. At the same time, the antigenic activity of virus nucleoprotein decreases perhaps due to the direct effect of tritium. The comparison of the degree of inactivation of various antigenic sites of the nucleoprotein within a virus labeled with 3H-uridine, suggests that they are located at different distances from RNA. A long-term action of 3H disintegration on RNA of a maturing virus decreases the yield probably due to the injury of the intracellular virus RNA during the infections process. Upon storage of the influenza virus labelled with 14C-amino acids the antigenic properties are reduced by the nucleoprotein while the infectiousness remains unaffected. The long-term effect of 14C disintegration on proteins of the maturing virus does not lead to fatal outcome.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive eLISA and for antigen capture in a sandwich eLISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from egyptian or South African lineages. these monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.Key words: RVF, nucleoprotein, competitive ELISA, RVFV lineages     

Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays

Monoclonal antibodies to five nonoverlapping antigenic domains of woodchuck hepatitis virus surface antigen (WHsAg) were used to develop site-specific radioimmunoassays. The assays were based on the solid-phase sandwich principle in which different combinations of individual domain-specific antibodies were used as immunoadsorbents and radioiodinated probes. Over 85% of the combinations tested were able to detect serum WHsAg, including those using the same antibody as immunoadsorbent and probe. The limits for antigen detection in one site-specific system ranged between 16 and 80 ng of WHsAg per ml. The antigenic similarity of serum WHsAg from 13 colony woodchucks was shown with several combination assay systems. WHsAg was equally immunoreactive in these assay systems whether obtained by immunoaffinity chromatography or standard rate zone centrifugation methods. Further site-specific analysis demonstrated that Formalin treatment of purified antigen did not affect the immunoreactivity of these WHsAg sites.     

Development, Characterization and Future Prospects of Monoclonal Antibodies Against Spores of Glugea atherinae (Protozoa-Microsporidia-Fish Parasites)

ABSTRACT. Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV' patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae . The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV⁺ patients.     

Study of the structural characteristics of the regulatory subunit of cAMP-dependent protein kinase type II using monoclonal antibodies

Monoclonal antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from porcine brain were used to study the antigenic properties of the enzyme regulatory subunit (RII). The monoclonal antibodies were bound to linear antigenic determinants on the protein molecule surface. The cAMP binding to RII interfered with the interaction between monoclonal antibodies and the protein. The use of different proteolytic fragments of RII allowed for the localization of antigenic determinants in the N-terminal moiety of RII.