CooA, which is a transcriptional regulator heme protein allosterically triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme in the Soret band is detected at approximately 1979 cm(-1), which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 and 290 ps, caused by the rebinding of the CO to the heme. About 98% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than those in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees on the basis of the anisotropy measurements. A shift of the bleached mid-IR spectrum of the bound CO is detected and has a characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in an active conformation and in an intermediate one, which is on the way toward an inactive conformation. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.
Bacterial Proteins , Hemeproteins/chemistry , Trans-Activators/chemistry , Carbon Monoxide/chemistry , Heme/chemistry , Hemoglobins/chemistry , Kinetics , Myoglobin/chemistry , Protein Conformation , Spectrophotometry, Infrared/methods
Titration of group B Salmonella O-antigen in the blood sera of patients and donors was carried out by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium LPS. Good correlation (r = 0.95) of the levels of S. typhimurium somatic O-antigen in the patients' sera determined by liposomal immunoassay and aggregate hemagglutination test was established. The concentration of the antigens in the tested samples was within 0.5-50 micrograms/ml. Statistical analysis of the results obtained by liposomal immunoassay techniques demonstrated differences in the distribution functions for the blood sera of patients with different diseases and of donors.
Antigens, Bacterial/blood , Lipopolysaccharides/blood , Salmonella Infections/immunology , Salmonella typhimurium/immunology , Adolescent , Child , Child, Preschool , Epitopes/blood , Humans , Immunologic Techniques , Infant , Influenza, Human/immunology , Liposomes , O Antigens , Pneumonia/immunology , Salmonella Infections/diagnosis
A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.
Antibodies, Bacterial/analysis , Antigens, Bacterial/analysis , Complement Hemolytic Activity Assay/methods , Salmonella typhimurium/immunology , Antibody Specificity/immunology , Dose-Response Relationship, Immunologic , Hemagglutination Tests , Humans , Lipopolysaccharides/immunology , Liposomes , O Antigens , Salmonella Infections/diagnosis , Solubility
The comparative evaluation of different immunological methods, such as the enzyme immunoassay, the aggregate hemagglutination test and the complement fixation test, used for the detection of specific Shigella antigens in biological body substrates obtained from 287 patients with acute dysentery caused by S. sonnei, S. flexneri and S. newcastle has been carried out. The enzyme immunoassay and the aggregate hemagglutination test most effective (97.5 +/- 0.5 and 92.4 +/- 0.9, respectively), the object of study being the patients' blood taken at the early stages of the disease. The diagnostic specificity of these methods has proved to be 98.7 +/- 6.7 and 95.2 +/- 1.4, respectively.
Antigens, Bacterial/analysis , Dysentery, Bacillary/diagnosis , Shigella flexneri/immunology , Shigella sonnei/immunology , Agglutination Tests , Complement Fixation Tests , Epitopes/analysis , Evaluation Studies as Topic , Hemagglutination Tests , Humans , Immunoenzyme Techniques , Salmonella Infections/immunology
Autoimmune Diseases/etiology , Immunization , Yersinia Infections/complications , Adult , Antibodies, Bacterial/analysis , Autoimmune Diseases/immunology , Chronic Disease , Cross Reactions , Humans , Immunoglobulin M/analysis , T-Lymphocytes/immunology , Yersinia/immunology , Yersinia Infections/immunology
Altogether 181 patients with typhoid and paratyphoid fever (54 with typhoid fever, 50 with paratyphoid fever type A, and 77 with paratyphoid fever type B) were investigated. Of them 108 (59.7%) patients were examined during the 1st week of disease. Serum specific O-antigens of typhoid and paratyphoid fever agents were determined by enzyme immunoassay (EIA), radioimmunoassay (RIA), and O-aggregate hemagglutination reaction (O-AHA), serum specific O-antibodies were determined by RDHA, EIA, RIA or O-AHA used during the 1st week of disease were twice as effective as RDHA. Combined use of EIA and O-AHA for the detection of serum specific O-antigens made it possible to diagnose typhoid fever in 90.91%, paratyphoid fever type A in 96.15%, and paratyphoid fever type B in 95.55% of cases.
Antigens, Bacterial/analysis , Paratyphoid Fever/diagnosis , Salmonella paratyphi A/immunology , Salmonella paratyphi B/immunology , Salmonella typhi/immunology , Salmonella/immunology , Typhoid Fever/diagnosis , Epitopes/analysis , Evaluation Studies as Topic , Hemagglutination Inhibition Tests , Hemagglutination Tests , Humans , Immunoenzyme Techniques , O Antigens , Radioimmunoassay , Time Factors
The solid-phase variant of radioimmunoassay for the determination of S. typhi O-antigen has been developed. The sensitivity of this method is 0.1 microgram/ml of the antigen in the blood serum of patients. The study of a number of blood serum samples collected from patients with typhoid fever has confirmed the possibility of using this method in clinical practice.
Antigens, Bacterial/analysis , Radioimmunoassay/methods , Salmonella typhi/immunology , Adsorption , Dose-Response Relationship, Immunologic , Evaluation Studies as Topic , Humans , O Antigens , Radioimmunoassay/instrumentation , Serologic Tests/instrumentation , Serologic Tests/methods , Time Factors , Typhoid Fever/diagnosis
The avidity of serum antibodies depending on infectious O-antigenemia in patients with paratyphoid B has been studied in the course of the disease. The study has revealed a faintly pronounced inverse correlation between the degree of avidity of serum antibodies and the level of infectious antigenemia. The avidity of serum antibodies in paratyphoid B has been found to increase in the course of the disease. A significant dependence of the severity of the disease on the avidity of serum antibodies has been noted.
Antibodies, Bacterial/analysis , Antibody Affinity , Antigens, Bacterial/analysis , Paratyphoid Fever/immunology , Salmonella paratyphi B/immunology , Salmonella/immunology , Hemagglutination Inhibition Tests/methods , Humans , O Antigens , Time Factors
The immunocorrecting agent levamisole decreases the duration of infectious O-antigenemia, determined in the O-aggregate hemagglutination test and in the enzyme immunoassay, essentially accelerates the progress of convalescence and considerably decreases the possibility of the prolonged relapsing course of the disease in comparison with common methods of treatment.
Antigens, Bacterial/analysis , Dysentery, Bacillary/drug therapy , Levamisole/therapeutic use , Adolescent , Adult , Drug Evaluation , Dysentery, Bacillary/immunology , Hemagglutination Tests , Humans , Immunoenzyme Techniques , Male , O Antigens , Recurrence , Shigella flexneri/immunology , Shigella sonnei/immunology
The analysis of serum samples from 124 patients with the bacteriologically confirmed diagnosis of group B salmonellosis has revealed that the specific neutralization variant of the enzyme immunoassay (EIA) makes it possible to detect IgA, IgG and IgM more effectively than the indirect EIA variant and the passive hemagglutination test.
Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Salmonella Infections/diagnosis , Evaluation Studies as Topic , Hemagglutination Tests/methods , Humans , Immunoenzyme Techniques , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Lipopolysaccharides/immunology , Neutralization Tests/methods , O Antigens , Salmonella typhimurium/immunology
A method for the statistical processing of data having different distribution functions is proposed. This method consists in the use of integral distribution functions. The description of the method based on the use of Soviet serial table-top microcomputers, model Elektronika MK-56, with specially developed programs (primary statistical data processing and single-factor regression analysis) is presented. The possibilities of this method are illustrated, by way of example, by processing the results of the determination of Shigella sonnei O-antigen, obtained by ELISA of serum samples taken from patients divided into different clinical groups according to the dynamics of the disease and the therapeutic methods used, as well as from control groups: healthy persons and patients with other intestinal diseases (salmonellosis and Escherichia coli infection). The above procedure has made it possible to differentiate the level of infectious O-antigenemia in accordance with the duration and the severity of the disease, as well as in accordance with the effectiveness of therapy.
Immunoenzyme Techniques , Adolescent , Adult , Antigens, Bacterial/analysis , Dysentery, Bacillary/immunology , Escherichia coli Infections/immunology , Humans , Methods , Microcomputers , O Antigens , Regression Analysis , Salmonella Infections/immunology , Shigella sonnei/immunology
The results of investigations carried out to study the effect of gamma radiation on the properties of the purified preparations of cholera exotoxin are presented. Irradiation has been shown to decrease the anterotoxicity of purified choleragen and the activity of its permeability factor, depending on the radiation dose. The investigations have revealed that in purified toxin enterotoxicity is completely inactivated with a lover radiation dose than in crude toxin filtrate (25 kGy). In immunochemical reactions the increase of the electrophoretic mobility of the choleragen components, correlated with the increase of the radiation dose, and the reduced number of protein zones have been observed. The irradiated preparations of purified choleragen have been found to retain their immunogenic properties and serological activity.
Cholera Toxin/radiation effects , Vibrio cholerae , Animals , Cholera Toxin/immunology , Cholera Toxin/isolation & purification , Dose-Response Relationship, Radiation , Electrophoresis, Polyacrylamide Gel , Gamma Rays , Immunochemistry , Immunodiffusion , Intestine, Small/drug effects , Precipitin Tests , Rabbits , Time Factors
The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied. This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments. The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy).
Enterotoxins/radiation effects , Exotoxins/radiation effects , Antigens, Bacterial/analysis , Antigens, Bacterial/radiation effects , Chemical Phenomena , Chemistry, Physical , Dose-Response Relationship, Radiation , Enterotoxins/immunology , Exotoxins/immunology , Gamma Rays , Immunochemistry , Immunologic Techniques
Crude cholera exotoxin (filtrate toxin) was irradiated with increasing doses of gamma radiation. A significant drop in enterotoxicity, in the activity of the permeation factor and a decrease in toxicity were shown to occur as radiation doses increased. Radiation doses of 50-70 kGy were found to completely inactivate enterotoxicity in liquid toxic preparations. A higher radioresistance of dried preparations in comparison with liquid ones was registered: inactivation occurred at 150-200 kGy. Different batches of the initial filtrate toxin had varying radiosensitivity. The sterilizing effect of gamma radiation was achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations. During the prolonged storage of the irradiated preparations of crude toxin (the term of observation being 1.5 years) at different temperatures no reversion of toxicity was found to occur, while their immunogenic properties remained unchanged.
Cholera Toxin , Exotoxins/radiation effects , Vibrio cholerae , Animals , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Exotoxins/immunology , Exotoxins/toxicity , Gamma Rays , Immunochemistry , In Vitro Techniques , Intestine, Small/drug effects , Mice , Rabbits , Skin/blood supply , Skin/drug effects , Toxoids/isolation & purification
