RESUMEN
The enzyme immunoassay (EIA) on nitrocellulose filters was adapted for the detection of exotoxin A in 104 P. aeruginosa strains isolated from patients and the environment in surgical wards in hospitals of Moscow and Alma-Ata. The method was shown to be highly sensitive: it permitted the detection of 5.ng of P. aeruginosa exotoxin A. The screening of 104 P. aeruginosa clinical strains by means of EIA on nitrocellulose filters revealed that these strains exotoxin A in 88.5% of cases.
Asunto(s)
Exotoxinas/análisis , Pseudomonas aeruginosa/patogenicidad , Colodión , Microbiología Ambiental , Filtración/instrumentación , Humanos , Técnicas para Inmunoenzimas/instrumentación , Kazajstán , Moscú , Pseudomonas aeruginosa/aislamiento & purificación , Servicio de Cirugía en HospitalRESUMEN
The diagnostic effectiveness of the passive hemagglutination (PHA) tests with the use of erythrocyte diagnostica (ED based on exotoxin A (ETA) and poly- and monovalent ED based on lipopolysaccharides (LPS) isolated from 5 most widespread P. aeruginosa serogroups was compared. 97 patients with different purulent septic diseases and 100 practically healthy adult donors were examined. The treatment of serum samples with 2-mercaptoethanol decreased the diagnostic effectiveness of serological examination. The simultaneous determination of the activity of antibodies to ETA and LPS essentially increased the number of positive results of the PHA tests in patients with bacteriologically confirmed P. aeruginosa infection, but not in patients from whom other bacteria were isolated. Considering the sensitivity and specificity of serological examination, as well as the decrease of the volume of necessary work and the consumption of immunoreagents, the optimum test conditions proved to be ensured by the use of two ED: LPS-based polyvalent ED and ETA-based ED.
Asunto(s)
Antígenos Bacterianos , Eritrocitos/inmunología , Pseudomonas aeruginosa/inmunología , Antígenos Bacterianos/inmunología , Estudios de Evaluación como Asunto , Exotoxinas/inmunología , Pruebas de Hemaglutinación , Humanos , Indicadores y Reactivos , Lipopolisacáridos/inmunología , Infecciones por Proteus/diagnóstico , Infecciones por Pseudomonas/diagnóstico , Pruebas Serológicas/métodos , Infecciones Estafilocócicas/diagnósticoRESUMEN
The protective properties of formulated toxoid obtained from the highly purified preparation of P. aeruginosa exotoxin A have been studied in the test of the active immunization of mice. The study has revealed that the preparation when introduced in 1 or 2 injections in a dose of 15 micrograms, shows faint protective potency with respect to P. aeruginosa strains differing in virulence. Immunization with this toxoid in 3 and 4 injections has been found to ensure 60-100% and 50-60% protection of mice infected with P. aeruginosa toxigenic and proteolytic strains respectively. Immunization with toxoid has been found to induce the appearance of short-term antibacterial immunity which loses its capacity to protect the immunized animals, challenged with both toxigenic and proteolytic P. aeruginosa strains, as early as on day 28. The immunization of mice with toxoid in 4 injections has been shown to induce the development of antitoxic immunity capable of neutralizing up to 150 LD50 of purified exotoxin A.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Exotoxinas/inmunología , Pseudomonas aeruginosa/inmunología , Toxoides/inmunología , Factores de Virulencia , Animales , Exotoxinas/aislamiento & purificación , Inmunización , Ratones , Toxoides/aislamiento & purificación , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The use of formulated chick red blood cells loaded with IgG preparations and affinity-purified antibodies, in comparison with initial immune serum to P. aeruginosa exotoxin A (ETA), has been shown to increase the sensitivity of antibody erythrocyte diagnosticum (AbED) 17-fold and to ensure the detection of ETA at a concentration of 1.2 mg of protein per ml. The passive hemagglutination (PHA) test with AbED has proved to be a more sensitive method for the detection of ETA than the antibody neutralization test with the use of antigenic erythrocyte diagnosticum, the latex agglutination test, the coagglutination test and the enzyme immunoassay. The PHA test has permitted the detection of ETA in the culture fluid of 80% of P. aeruginosa cultures under study.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Eritrocitos , Exotoxinas/análisis , Pseudomonas aeruginosa/metabolismo , Factores de Virulencia , Medios de Cultivo , Humanos , Sueros Inmunes , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The use of polyvalent erythrocyte diagnosticum prepared on the basis of 5 polysaccharide antigens of P. aeruginosa slime, isolated from strains belonging to the most widespread serovars, makes it possible to check up the humoral response of donors after their immunization with P. aeruginosa polyvalent corpuscular vaccine with the aim of obtaining anti-P. aeruginosa donor plasma. Antibody titers, determined in the passive hemagglutination test with the use of the proposed diagnosticum and corresponding to a serum dilution of 1:320 and greater, age tentatively diagnostic, which may be indicative of P. aeruginosa in the development of purulent septic complications in patients. The use of the passive hemagglutination test with the newly developed polyvalent erythrocyte diagnosticum makes it possible to check up the specific response of patients having P. aeruginosa infection in the process of their treatment with anti-P. aeruginosa hyperimmune plasma used as a part of complex therapy.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Eritrocitos/inmunología , Infecciones por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/inmunología , Especificidad de Anticuerpos , Vacunas Bacterianas/inmunología , Donantes de Sangre , Quemaduras/inmunología , Pruebas de Hemaglutinación/métodos , Humanos , Heridas y Lesiones/inmunologíaRESUMEN
Specific antibodies for immunoenzymatic assay of P. aeruginosa exotoxin A were isolated with affinity chromatography on exotoxin A-Sepharose 4B. A conjugate of the antibodies to exotoxin A with beta-lactamase from Bacillus licheniformis 749/c was prepared by linking glutaric aldehydes. The conjugate showed high immunospecific and enzymatic activity and was used for competitive solid phase immunoenzymatic assay. Sensitivity of the assay provided determination of exotoxin A in concentration of 20 ng/ml. It was used for determining P. aeruginosa exotoxin A in culture fluids.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Exotoxinas/análisis , Técnicas para Inmunoenzimas , Pseudomonas aeruginosa , Factores de Virulencia , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Especificidad de Anticuerpos , Bacillus/enzimología , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Exotoxinas/aislamiento & purificación , Inmunización , Pseudomonas aeruginosa/inmunología , Conejos , beta-Lactamasas , Exotoxina A de Pseudomonas aeruginosaRESUMEN
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).
Asunto(s)
Antígenos Bacterianos/inmunología , Eritrocitos/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Relación Dosis-Respuesta Inmunológica , Pruebas de Hemaglutinación , Humanos , Inmunización , Infecciones por Pseudomonas/diagnóstico , ConejosRESUMEN
The present investigation has revealed the possibility of using different kinds of monodispersed polystyrene latex, produced in the USSR, as carriers in the process of the preparation of antibody diagnosticums intended for the detection of water-soluble slime antigens of Pseudomonas aeruginosa strains belonging to the most widespread serological types. The optimum conditions for the preparation of latex reagents and for making the latex-agglutination test have been experimentally established. The new diagnosticums+ have been shown to be highly species- and type-specific, which permits making judgment on the presence or absence of slime antigens of P. aeruginosa strains belonging to definite serovars in the clinical material under study. The preparations thus obtained have been found to retain their sensitivity for 16 months (the term of observation).
Asunto(s)
Pruebas de Fijación de Látex/métodos , Infecciones por Pseudomonas/diagnóstico , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/análisis , Estudios de Evaluación como Asunto , Humanos , Indicadores y Reactivos , Látex , Tamaño de la Partícula , Pseudomonas aeruginosa/inmunología , Solubilidad , Especificidad de la EspecieRESUMEN
The possibility of using antigenic complexes contained in the extracellular slime of P. aeruginosa clinical strains belonging to different serological groups as the components of a chemical vaccine has been revealed. Animal experiments have demonstrated a high immunogenicity of these preparations, as well as their low toxicity. The use of slime antigens stimulates the production of specific antibodies exerting a protective action against infection with homologous P. aeruginosa strains.