Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells.

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

[Alterations in heat shock protein 70 kDa levels in human neutrophils under the heat shock conditions].

Intracellular content of heat shock proteins of 70 kDa family (HSP70) possessing chaperone and cytoprotective functions depends on specialization and functional activity of the cells. The aim of this study was to analyze the dynamics of constitutive and inducible HSP70 levels evoked by heat shock in human neutrophils, the short-lived fraction of white blood cells providing non-specific defense against bacterial pathogens. Biphasic dynamics of the intracellular HSP70 level with an increase and following decrease in 15-30 min after the heat shock was revealed by flow cytometry. This dynamics was similar for constitutive and inducible forms of HSP70. Pre-incubation of neutrophils with cycloheximide, the inhibitor of protein synthesis, did not change the intracellular HSP70 dynamics registered by flow cytometry indicating that the increased HSP70 level detected immediately after the heat shock was not mediated by de novo protein synthesis. It was confirmed by Western blotting analysis of HSP70 intracellular content. It was suggested that the elevated HSP70 level was related to conformational HSP70 molecule changes and to increased availability of HSP70 epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or C-terminal substrate-binding domains of HSP70 it has been demonstrated by cell immunofluorescence and flow cytometry methods that the heat shock-associated increase of the intracellular HSP70 level was mediated by HSP70 interaction with antibodies recognizing HSP70 substrate-binding domain. It was demonstrated that the decrease of intracellular HSP70 level after heat treatment could be connected with a release of both inducible and constitutive HSP70 into extracellular space. Our data suggest that stress-induced release of HSP70 from neutrophils is regulated by ABC-transporters.

[Genetic and immunochemical study of Legionella strains isolated in Sverdlovsk region].

Two strains of Legionella isolated from patient and hot water supply system during outbreak in Sverdlovsk region in 2007 were studied. Using genetic analysis methods (genes sequencing, VNTR-typing and PCR-based study of omp 28 gene), it was shown that tested strains are pathogenic but do not belong to one genetic group and epidemic cluster. Performed immunochemical analysis confirmed that both isolated strains belong to Legionella serogroup 1.

Monoclonal antibodies directed against lipopolysaccharide of Legionella pneumophila serogroup 1.

The monoclonal antibodies (MAbs) against lipopolysaccharide of virulent strain of Legionella pneumophila serogroup 1 were produced. Three most productive hybrid clones (5F4, 5F10 and 2C9) were selected from fusions of mouse myeloma cells with spleen cells from BALB/c mice, immunized with bacterial outer membrane antigens. All generated clones were IgG-secreting. The MAbs had narrow strain specificity and showed no cross-reactions with other unrelated bacterial species. These antibodies were tested in sandwich ELISA. The results suggest that the MAbs could be used for diagnostic purposes.

[The determination of the antigenic determinant of protective monoclonal antibodies specific to the Francisella tularensis lipopolysaccharide]. / Opredelenie antigennoi determinanty preventivnykh monoklonal'nykh antitel, spetsifichnykh k lipopolisakharidu Francisella tularensis.

F. tularensis lipopolysaccharide (LPS) was studied with the use of monoclonal antibodies (McAb) having protective properties. The binding site of these McAb (IgG2a) is localized on the O-chain of LPS. In contrast to LPS isolated from vaccine strain 15, LPS isolated from F. tularensis cells in the R-form has no O-chains and does not interact with McAb.

[The preventive activity of monoclonal antibodies specific to the lipopolysaccharide of Francisella tularensis]. / Preventivnaia aktivnost' monoklonal'nykh antitel, spetsifichnykh k lipopolisakharidu Francisella tularensis.

The preventive activity of five monoclonal antibodies (McAb) in experimental tularemia was evaluated. McAb produced by hybridoma FB11-k (IgG2a), specific to F. tularensis lipopolysaccharide, prevented the death of mice and guinea pigs infected with F. tularensis virulent strain 503 of the holarctic subspecies.

[Concentration of cyclic nucleotides, activity of adenylate cyclase, 3',5'-AMP phosphodiesterase and guanylate cyclase in plasma membranes from liver and hepatomas of different degrees of malignancy]. / Soderzhanie tsiklicheskikh nukleotidov, aktivnost' adenilattsiklazy, fosfodiésterazy 3',5'-AMF i guanilattskilazy v plazmaticheskikh membranakh pecheni i gepatomakh razlichnoi stepeni zlokachestvennosti.

Adenylate cyclase activity as well as intracellular content of sAMP were decreased 2.5-4-fold, as compared with normal state, in plasmatic membranes (PM) of hepatoma 22 and of Ehrlich ascites carcinoma--the tumors characterized by high level- of malignancy. Activity of cAMP phosphodiesterase exceeded distinctly the normal value in all the tumors studied. In less malignant hepatoma 48 the adenylate cyclase activity and content of cAMP were similar to those found in normal liver cells. The guanylate cyclase activity did not differ markedly from values found in normal liver cells in PM of all the tumors studied and in liver tissue of the tumor-bearing animals. Distinct alterations were not found in content of cGMP in the tumors, except of hepatomas 60 and 22, in which the nucleotide level exceeded 2-fold the normal value. The ratio cAMP/cGMP was decreased in the most malignant tumors. At the same time, the ratio was distinctly elevated in tumors with the middle level of malignancy (hepatomas 60 and 61).

[Adenosine deaminase activity in hepatomas of varying degrees of malignancy]. / Aktivnost' adenozindezaminazy v gepatomakh razlichnoi stepeni zlokachestvennosti.

In highly malignant Gelshtein 22A hepatcma and ascites Ehrlich carcinoma adenosine deaminase activity was found to be reduced 3-fold as compared with that of the normal mouse liver. In less malignant hepatomas adenosine deaminase activity drops only by 20%. A certain reduction of adenosine deaminase activity was also noted in the liver to tumour-bearing mice.