Development of a polyvalent erythrocyte diagnostic agent based on the antigens of Pseudomonas aeruginosa slime

The study has revealed the fundamental possibility, and established the concrete conditions of obtaining standard erythrocyte polyvalent diagnosticum for the detection of antibodies to the antigens of P. aeruginosa, belonging to 5 types most frequently occurring in clinical practice, in the passive hemagglutination test. The data on high type- and species-specificity of the new erythrocyte diagnosticum have been obtained, which indicates that slime antigens have been correctly chosen as sensitins and confirms the necessity of using the polyvalent preparation. Preliminary data on the clinical trial of the erythrocyte diagnosticum show the expediency of its use for the control of specific immune response in donors in the process of obtaining anti-P. aeruginosa hyperimmune plasma, as well as the possibility of using this method for the serological diagnosis of P. aeruginosa infection. The diagnostic preparation has been found to retain its activity for 10 months (the term of observation).     

Production of an diagnostic erythrocyte agent from Pseudomonas aeruginosa exotoxin A

The sensitization of formolized sheep red blood cells with exotoxin A by means of chromium chloride or glutaraldehyde is more effective with respect to their sensitivity in the passive hemagglutination test than loading by means of amidol, tannin and rivanol. The use of chromium chloride decreases the consumption of exotoxin A 2, 8, 16 and 16 times in comparison with the use of amidol, tannin, rivanol or glutaraldehyde respectively. The high specificity of erythrocyte diagnosticum obtained from exotoxin A by means of chromium chloride is indicated in the study of hyperimmune sera to 22 different antigens of enteric bacteria and staphylococci in the passive hemagglutination test and to 10 different enterobacterial and staphylococcal antigens in the antibody neutralization test.     

Use of monoclonal antibodies for the production of a plague antibody erythrocyte diagnostic agent

Monoclonal antibodies to Yersinia pestis capsular antigen were fixed onto the surface of formulated sheep red blood cells. The preparation thus obtained was compared with commercial antibody erythrocyte diagnosticum in the passive hemagglutination test aimed at the search for the capsular antigen in the suspensions of Yersinia pestis museum cultures and in the antigen neutralization test aimed at the search for antibodies in the sera of wild and laboratory animals having had plague. Monoclonal erythrocyte diagnosticum proved to be suitable for the detection of both the capsular antigen and antibodies. The comparison of the results of the passive hemagglutination test and the enzyme immunoassay demonstrated the presence of very close relationship between them.     

Trial of an erythrocyte antigenic diagnostic agent for detecting antibodies to Coxiella burnetii

A new method for the preparation of Q-fever erythrocyte antigenic diagnosticum, adaptable to large-scale production, has been developed, and the diagnosticum thus obtained has been found to be highly specific and sensitive. The combined use of the complement fixation and passive hemagglutination tests enhances the effectiveness of the serological study, as it not only ensures a more complete detection of antibodies to C. burnetii, phase I, in humans and cattle, but also gives more precise indications on the time of infection.     

Principle of constructing polyvalent Pseudomonas aeruginosa vaccines]

Dependence of the range of protective action of P. aeruginosa vaccine on the number of its composites was studied. A principle of the selection of strains who vaccines differed in vivo by immunological specificity was applied to construction of the experimental preparations and modelling a polyvalent vaccine. Increase of the number of components in the vaccine was accompanied by increase of its protective action range. However, with the increase of the number of polyvaccine components in the polyvaccine the accretion of the protective effect expressed in the mean protective index per component displayed a gradual reduction. It was calculated theoretically that a 6--7-component vaccine should provide protection from 94--96% of the P. aeruginosa strains; as to further increase of the number of components--it would induce overloading of the vaccine with a possible absence of any effect.     

Effectiveness of a polyvalent corpuscular Pseudomonas aeruginosa vaccine, antibiotics and their combinations in experimental chronic Pseudomonas aeruginosa infection

The data on the development of the experimental model of P. aeruginosa chronic infection in mice, produced by their intraperitoneal inoculation with the infective agent, and on the study of the properties of this model are presented. The model has been used in the experimental study of the preventive action of P. aeruginosa polyvalent corpuscular vaccine. The comparative study, carried out with the use of the proposed model, has been made with a view to evaluating the effectiveness of different methods for the treatment of P. aeruginosa chronic septic infection by means of antibiotics (polymixin B and tobramycin), P. aeruginosa polyvalent corpuscular vaccine and their combination. The combined use of this vaccine with antibiotics (polymixin B or tobramycin) has proved to give the most pronounced curative effect with respect to P. aeruginosa chronic infection.     

Isolation and study of the properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine. An evaluation of the biological properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine

P. aeruginosa killed polyvalent corpuscular vaccine, tested in a trial on 42 volunteer donors, is safe, low reactogenic and possesses pronounced immunogenicity, as the injection of the vaccine induced a rise in the titer of antibodies to P. aeruginosa up to 1:1280 in 95% of the immunized donors. To obtain specific antibodies in the blood plasma of the donors in an amount sufficient for the protective activity of the plasma becoming manifest, it is expedient to use the vaccination schedule providing for subcutaneous injections of 0.5, 0.5 and 1.0 ml at intervals of 7 days. Intense immunity thus induced in the donors lasts for 3-4 months. The hyperimmune plasma obtained from the donors has an antibody titer of at least 1:320 and shows a 90-100% protective effect in mice infected intraperitoneally with P. aeruginosa. The preliminary results of the clinical trial of anti-P. aeruginosa plasma have demonstrated its efficiency as a part of the complex treatment of patients with purulent septic complications of P. aeruginosa etiology.     

Protective effect of a corpuscular polyvalent Pseudomonas aeruginosa vaccine in generalized chronic Pseudomonas aeruginosa infection in mice with cyclophosphamide-induced leukopenia

The prophylactic effect of immunization with P. aeruginosa polyvalent corpuscular vaccine has been shown on the model of P. aeruginosa generalized chronic infection in mice with leukopenia induced by the intraperitoneal injection of cyclophosphamids. This effect is manifested by the increased resistance of the animals to sublethal doses of P. aeruginosa strain, as well as by more intense general and specific immunological responses in the infected animals (the increase of specific antibody titers, the number of leukocytes in the blood serum and the phagocytic activity of the cells of peritoneal exudate).     

Protective action of a corpuscular polyvalent Pseudomonas aeruginosa vaccine against representative enterobacteria

Animal experiments have demonstrated that P. aeruginosa vaccine is capable of protecting animals from experimental P. aeruginosa infection, as well as rendering a protective effect with respect to some representatives of the family Enterobacteriaceae. The comparative study of the antigenic spectra of the vaccine strains and some representatives of Enterobacteriaceae (Enterobacter, Serratia, Citrobacter and Klebsiella) has revealed no direct relationship between the degree of this protective effect and the presence of common antigenic determinants in them.     

Obtaining antisera to Pseudomonas aeruginosa and Proteus antigens for performing immunoenzyme analyses in clinical practice

The data presented in this work indicate that specific antisera to P. aeruginosa and Proteus antigens can be produced by using extracts from these microorganisms, destroyed by ultrasonic treatment or by multiple freezing and thawing, for the immunization of rabbits. Blood serum samples from patients with purulent septic complications were studied for the presence of P. aeruginosa and Proteus antigens in ELISA with the use of peroxidase-labeled antibodies from antisera to P. aeruginosa and Proteus. This investigation revealed that during the first 3 days from the beginning of the clinical manifestations of the complications P. aeruginosa and Proteus antigens were detected in 86.4% and 83.4% of the patients, respectively. In the subsequent bacteriological study of wound discharge from these patients the corresponding microflora was detected.     

Effectiveness of the tularemia-antibody erythrocyte diagnostic agent for the detection of specific antigen and antibodies to it]

The authors demonstrated the efficacy of utilization of tularemia antibody erythrocytic diagnostic agent. Data are presented indicating a strict specificity and a high sensitivity of this diagnostic agent for detection of the Vi-antigen both in the tularemia microbe cultures and in the suspensions of the organs of rodents which perished of tularemia, irrespective of the state of cadaver. There was revealed relationship between the sensitivity of the diagnostic agent and the virulence of the tularemia microbe cultures. A possibility of using the diagnostic agent in the antigen neutralization test for detection of specific antibodies in the sera of patients, who sustained the disease, and of the vaccinated humans and animals was revealed.     

Use of the pneumococcal serovar 3 erythrocyte antibody diagnostic agent for analyzing polysaccharide-containing preparations

Antibody diagnosticum to pneumococci (serovar 3) has been prepared. The analysis of different antigenic preparations of pneumococci (serovars 1,3 and 6B) and the filtrates of culture fluid obtained in the process of the cultivation of bacteria belonging to serovars 3,9N and 23F has been carried out by means of the indirect hemagglutination test. The new diagnosticum has proved to be type-specific. This diagnosticum can be used for the evaluation of the quantitative content of specific polysaccharide in different antigenic preparations of serovar 3 pneumococci, as well as for the determination of the content of specific polysaccharide directly in the culture fluid, which constitutes a step in the determination of the period of the maximum synthesis of polysaccharide, necessary for the optimization of the process of the cultivation of pneumococci.     

Natural antibodies in Pseudomonas aeruginosa infections

Studying 347 sera of 49 patients suffering from Pseudomonas aeruginosa infection the mean titre value (MTV) calculated from the exponent of the binary logarithm of natural antibody (NA) titres, was found suitable to characterize the humoral immune status. As long as the organism is in equilibrium with the infection, the NA level rises. In septic shock, before death or at the development of a massive infection, the NA titre decreases rapidly. The decrease may be due partly to the permeability increasing effect of endotoxin and antigen-antibody reactions exerted on the capillaries. Consequently, in the most severe phase of sepsis, when bacteria enter the circulation less NA is available to fight against them. This might be a cause of the still very high lethality of septic shock due to Gram-negative bacteria. Finally, when applying the MTV one always has to consider that despite its advantages it gives less information than the Backhausz immunogram.     

Use of immunosorbents for determining antibodies to histaglobulin]

A determination was made of antibodies to histaglobulin and gamma-globulin with the aid of immunosrobents in 178 sera of children suffering from allergic diseases, during the histaglobulin therapy. Results of investigations showed antibodies to histaglobulins to be absent in children untreated by this preparation. But they appeared in 57% of cases after the first course of histaglobulin treatment, and their incidence and their average level increased with an increase in the number of the courses of treatment carried out. gamma-Globulin antibodies were found at the initial condition in 42% of cases; this percentage rose to 68 after the first course of histoglobulin treatment. The authors believe that determination of histaglobulin antibodies during the treatment with this preparation could serve as an auxiliary immunological criterion of the efficacy of histaglobulin therapy.     

Erythrocytic diagnostic agents for determining antibodies to Proteus O antigens

Highly sensitive and specific erythrocyte diagnostic agents (ED) for the determination of antibodies to Proteus O-antigens have been obtained by the sensitization of formolated sheep red blood cells (SPBC) with activated lipopolysaccharides (LPS) without the use of mediators. The tannin treatment of formolated SRBC and/or the increase of temperature from 45 degrees C to 100 degrees C in the process of the preparation of ED have been found to produce no increase in effectiveness. Antibody ED permitting the detection of Proteus O- and H-antigens has been obtained by the sensitization of formolated chick red blood cells with immunoglobulin preparations to Proteus hydroxylamine antigens, carried out with the use of amidol. The experiments have shown the possibility of using this antibody ED for the determination of O-antibodies in the antigen neutralization test with nonactivated LPS used as an agglutinating agent. The passive hemagglutination test with antibody ED has proved to be a more sensitive method for the detection of O-antibodies than the antigen neutralization test with antigenic ED. The determination of Proteus etiology in the passive hemagglutination test with the use of antigenic ED has been shown to be highly effective in the examination of patients with chronic osteomyelitis at the stage of exacerbation.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

New selective agent for isolation of Pseudomonas aeruginosa.

Results of minimal inhibitory concentration tests with a diversity of bacterial strains showed that 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390) inhibited the growth of all microorganisms tested (other than Pseudomonas aeruginosa) at 25 microgram/ml or less, whereas MICs obtained for P. aeruginosa ranged from to to greater than 100 microgram/ml. Therefore, C-390 was evaluated as a potential selective agent for P. aeruginosa in pseudomonas agar F. Recovery tests were conducted on this medium with 53 strains o P. aeruginosa, and the results were compared to those obtained in similar tests on commercially available selective media, i.e., pseudomonas isolation agar and Pseudosel agar. The results of these comparisons indicated that pseudomonas agar F with C-390 was significantly less inhibitory than Pseudosel agar and pseudomonas isolation agar and more selective than pseudomonas isolation agar. The incorporation of C-390 in pseudomonas agar F also provided a medium that was both selective and differential. Preliminary evidence also suggested that C-390 may be added to other basal media with comparable results.