Effect of detoxification processes on the interferon-inducing activity of bacterial endotoxins.

The interferon (IFN)-inducing activity of detoxified lipopolysaccharide (LPS) was tested in rabbits treated with LPS preparation derived from Escherichia coli, Salmonella typhi, Salmonella enteritidis and Shigella dysenteriae serovar 1. Of the detoxification procedures used, alkaline hydrolysis, hydroxylaminolysis, formalization, treatment with sodium deoxycholate and the radiodetoxification (fast or slow) methods had no appreciable effects on the IFN-inducing potential of LPS. In contrast, acetylation or prolonged alkaline hydrolysis of LPS resulted in up to a 9-fold reduction of IFN-induction capacity and effects of Cu++ or Fe++ cations bound to LPS were clearly inhibitory (Fe more than Cu).

Preparation and properties of monoclonal antibodies against Mycobacterium tuberculosis.

The above mentioned monoclonal antibodies of idiotype IgG 3/Kappa against TMC 120 and TMC 107 strains of M. tuberculosis were prepared by fusion of mouse myeloma line FO cells with splenocytes BALB/c of mice immunized with complex antigens. These were prepared from the bacterial mass previously inactivated with gamma-rays radiation, and applied without mycobacterial adjuvant agent. Species specificity of both monoclonal antibodies was determined by ELISA testing and dot blot test. The potential use of the mentioned preparations consists in species identifying and taxonomy of mycobacteria, mycobacterial antigen purification and prompt TBC diagnosis (serodiagnosis, identifying of specific antigens in pathological materials.

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization.

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.

A monoclonal antibody to Mycobacterium kansasii: preparation and properties.

An IgG2/kappa monoclonal antibody specific for a subunit protein antigen (mol.w. 32 KD) obtained by means of gel chromatography of the cell-free sonicate of M. kansasii was generated by the fusion of spleenocytes from BALB/c mice immunized with this antigen and Ag-8 myeloma cells. Its specificity was demonstrated by ELISA and dot blot procedures. This MoAb has potential application in taxonomy and species identification of M. kansasii isolates, in studies relating to the molecular pattern of the bound antigen and in serodiagnostics of the mycobacterial disease due to M. kansasii.

Inactivation by polymyxin B of the endotoxin-mediated interferon production in the rabbit.

Polymyxin B (PB) completely or at least significantly inhibited the capacity of Shigella dysenteriae 1 cells and the lipopolysaccharide (LPS) and lipid A (LA) subunits of several bacterial endotoxins to induce interferon (IFN) in rabbits. Animals injected with LPS inactivated by PB to the point of not inducing detectable IFN levels did not develop hyporesponsiveness to secondary IFN induction by a homologous inducer. It was concluded that PB inhibits the IFN-inducing capacity of endotoxin and its subunits as a consequence of binding to the LA-moiety of LPS. The results confirmed the exclusive role of LA as the only IFN-inducing component of Gram-negative bacterial endotoxin.

Determination of infectivity of transforming avian sarcoma virus and parotitis virus in fibrin-embedded monolayer cultures.

A sensitive and reproducible technique for titration of transforming avian sarcoma virus, Rous sarcoma virus (RSV), in chick embryo cell monolayers embedded between two layers of a solid fibrin coagulum was developed. The foci of RSV-transformed fibrinolysis-exhibiting cells locally attacked the surrounding fibrin and were identifiable as plaque-like cell-free areas of retraction easily scored against the background of cells stained with neutral red. In comparison to the conventional assay in agar, the assay in fibrin proved more sensitive and less time-consuming. The titration technique in fibrin was also used for plaquing a cytopathogenic virus, parotitis virus, but the titration by conventional plaque assay in agar and the technique in fibrin yielded similar results in this case.