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).     

Erythrocyte diagnostic kits for detection of Pseudomonas aeruginosa exotoxin A

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.     

Antibody response to capsular polysaccharide antigen in an infection caused by meningococcus serogroup B in children

The continuous synchronous cultivation of serogroup B meningococci and the use of the selected clones of producer strains has made it possible to obtain the serologically active preparation of the group-specific antigen from the culture in the exponential phase. The erythrocyte diagnosticum produced on the basis of this preparation has permitted the detection of group-specific antibodies in the sera of immunized rabbits and children with generalized infection caused by serogroup B meningococci. The intensity of immune response depends on the age of children. This new technique of the passive hemagglutination test is recommended for the confirmation of generalized infection caused by serogroup B meningococci.     

Serological diagnosis of suppurative-septic diseases of staphylococcal etiology

The parallel examination of osteomyelitis patients by means of the passive hemagglutination (PHA) test with the use of antigenic erythrocyte diagnostic agents, prepared on the basis of hydrochloric acid extract from Staphylococcus aureus strain 209P and teichoic acid extract from S. aureus strain Wood 46 has revealed that these diagnostic agents are practically equal in their diagnostic effectiveness. In the examination of endocarditis patients measurement of the total antibody activity in the PHA test with diagnosticum on the basis of strain 209P has proved to be a more sensitive method, whereas in osteomyelitis patients the total IgG activity has been more accurately measured by ELISA. The treatment of sera with 2-mercaptoethanol essentially decreased the diagnostic effectiveness of the PHA test. The study has shown the diagnostic value of not only IgG but also of IgM antibody measurements.     

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.     

The diagnostic value of a meningococcal-B erythrocyte diagnostic agent based on the group-specific polysaccharide

In this work the diagnostic value of group B meningococcal erythrocyte diagnosticum was determined. 585 blood serum samples taken from adult donors were studied: 220 samples from practically healthy persons and 365 samples from 144 patients with meningococcal infection and purulent bacterial meningitis of nonmeningococcal etiology. Group B meningococcal erythrocyte diagnosticum was found to possess serological activity and to reveal the growth of specific antibodies in the sera of patients with meningococcal infection, serologically confirmed by the isolation of group B meningococcal culture, in 100% of cases on weeks 2-3 of the disease. Diagnostic characteristics--specificity and sensitivity--for group B erythrocyte diagnosticum were, respectively, 90.2% and 63.5%. The study revealed that antibodies to several group-specific meningococcal polysaccharides in blood sera can be simultaneously determined in the passive hemagglutination test with a set of erythrocyte diagnostica, which should be taken into consideration in the clinical interpretation of serological results.     

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.     

Obtaining erythrocyte diagnosticum for the detection of chlamydial antigen

It is shown possible to obtain erythrocyte diagnosticum for detection of chlamydial antigen, whose cell basis consists of a formalinized suspension of ram erythrocytes, sensibilized with hyperimmune antichlamydial sera by means of the amydol. High sensitivity and specificity of the diagnosticum, absolute correlation with the data obtained in the complex examination of patients with the urogenital tract pathology of the chlamydial etiology by other methods were determined in the course of investigation in the indirect hemagglutination test with diagnosticums of scrape specimens from these patients.     

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.     

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.     

The results of a controlled field trial of a dried erythrocytic immunoglobulin diagnostic agent for detecting the hepatitis A virus antigen]

The results of the controlled field trial of lyophilized erythrocytic immunoglobulin diagnosticum for the detection of hepatitis A virus antigen in the urine and feces of patients are presented. This diagnosticum was used for the study of urine and fecal samples from 225 patients (of these, 176 had hepatitis A) and 54 healthy persons in the passive hemagglutination (PHA) test. Their blood sera were studied in the PHA test (to detect HBsAg) and the radioimmunoassay (to detect anti-HAV IgM). The immunoglobulin diagnosticum under study was found to be nonspecific and faintly sensitive and, therefore, unsuitable for use in medical practice.     

A comparative evaluation of the sensitivity of different methods for the serological diagnosis of leptospirosis

In this work the comparative evaluation of the sensitivity and serological specificity of the microcapsular agglutination (MCA) test, the passive hemagglutination (PHA) test and the microagglutination (MA) test are presented. In the MCA test leptospiral antigens, adsorbed on synthetic carrier capsules produced by Japan Lyophilization Laboratory, were used and the PHA test was made with the use of polyvalent erythrocyte diagnosticum. The study of blood serum samples from 46 leptospirosis patients revealed that the values of antibody titers in the PHA and MCA tests were 5.5-8.1 times higher than the traditional MA test. In the MCA and PHA tests antileptospiral antibodies could be detected as early as on days 1-3 of the disease when the results of the MA test were negative or very low. The maximum values of antibody titers in the MCA and PHA tests were detected on days 11-15 of the disease and in the MA test, on days 21-25. The MCA and PHA tests are genus-specific and permit the detection of antileptospiral antibodies irrespective of the serogroup of the infective agent. In the study of the blood sera of 40 patients with diseases of nonleptospiral etiology the MCA and MA tests yielded false positive results in 7.5% and the PHA test, in 12.5% of cases in titers below the diagnostic level. These data are indicative of high sensitivity and specificity of the serological tests used in this study.     

Serological diagnosis and immunological aspects of Proteus infection. V. Design and trial of polyvalent antigenic preparations

The main principles of the development of Proteus polyvalent antigen and erythrocyte diagnosticum, including the selection of the initial strains according to the clinical importance of their H-antigens and to the variety of their partial factors, the combination of monoantigens to form a polyvalent antigenic preparation with due regard to the potency of each component and the use of the preparation in serological tests in accordance with its summary potency, as well as the simultaneous loading of formaldehyde-fixed and tannin- or bisdiazo benzidine-treated sheep red blood cells with sensitins, have been worked out. The diagnostic value of these preparations has been confirmed by the study of serum samples from 579 patients with intestinal and urological infections (among them 153 patients releasing Proteus) and 245 healthy persons.     

Escherichia-erythrocyte diagnostic agents

The conditions of a simple and practicable method for the preparation of effective antigenic nonprotein diagnosticums on the basis of water-phenol extracts of 23 Escherichia species have been developed. The method consists in heating the mixture of erythrocytes and the antigen in a boiling water bath for 60 minutes. The diagnosticums thus obtained are 16-30 times more sensitive in the passive hemagglutination test and 4-6 times more sensitive in the passive hemagglutination inhibition test than diagnosticums prepared with the use of tannin, rivanol, as well as by the common method for the preparation of nonprotein antigens. The minimum concentration of Escherichia cells detected in the passive hemagglutination inhibition test is 0.8-1.2 million cells/ml.     

Pili (fimbriae) of the causative agents of plague and pseudotuberculosis: their detection in the hemagglutination reaction, the passive hemagglutination reaction and in an immunoenzyme analysis system

Newly developed serological methods for the detection of pili in the passive hemagglutination (PHA) test with the use of immunoglobulin erythrocytic diagnosticum and in the enzyme immunoassay (EIA) with the use of specific immunoglobulins labeled with horse radish peroxidase have been found to exceed the method of detecting pili, based on the determination of their hemagglutinating activity, in sensitivity and specificity. Besides, the PHA test and EIA have proved to be capable of detecting low molecular fragments of pili, obtained by sonication and having lost their hemagglutinating activity.     

Cross-reacting intraspecific antigens of Neisseria meningitidis. I. The isolation, immunochemical and serological characteristics of the cross-reacting antigens of N. meningitidis serogroup A

New data on the cross-reacting antigen of N. meningitidis, serogroup A, are presented. A complex of antigens has been isolated by treatment with tryptone X-100, ethanol precipitation and the subsequent treatment with trichloroacetic acid. The immunological analysis of the isolated preparation has shown that the proteinaceous part of the biopolymer contains 7 polypeptide fragments; of these, one fragment with a molecular weight of 31000 daltons has been found to constitute 49, 15% of all polypeptide fragments. The evaluation of the serological properties of this preparation in the precipitation test and the passive hemagglutination test has revealed that the preparation contains various cross-reactive antigenic determinants. Polyvalent erythrocyte diagnosticum obtained on the basis of this preparation permits the detection of antibodies to meningococci irrespective of their serogroup.     

The improvement of the immunodiagnosis of para-influenza infection based on erythrocyte reagents]

Study of different methods of hemosensitization helped detect the optimum technology for the preparation of parainfluenza (type 3) antigenic erythrocyte diagnosticum. This technology ensures the highest sensitivity, specificity, economy (with respect to viral antigen consumption) and stability of the reagent. Titration of antibodies by means of the new diagnosticum and the detection of parainfluenza cases among children with acute respiratory virus infections, acute and chronic glomerulonephritis have been highly effective, considerably more effective than similar determinations by the hemagglutination inhibition test.     

Serologic diagnosis of intestinal yersiniosis. The design of a stable erythrocyte preparation for the indirect hemagglutination reaction

Nine types of erythrocyte diagnostica of serovars O3 and O9, differing in the methods of obtaining sensitins and the physical state of erythrocytes, were put on trial. The preparations were used for the titration of hyperimmune sera and blood sera obtained from about 500 healthy persons, 300 patients with Yersinia enteric infection and 300 patients with other diseases. Freeze-dried diagnostica, when compared with liquid ones, were found to be less sensitive, but more stable and specific. Sensitins isolated by the methods of Westphal ad Boivin showed the highest degree of specificity. The authors believe freeze-dried sheep red blood with activated Boivin's antigen adsorbed onto them to be the optimal preparation for use in the passive hemagglutination (PHA) test. The preparation was found to retain its serological activity for as long as 2-3 years. The titer 1:160 (1:200) in the PHA test is recommended as the minimal diagnostic indicator. Erythrocyte diagnosticum is more sensitive, specific and stable than bacterial one. Since 1984 dried Yersinia erythrocyte diagnostica (serovars O3 and O9) have gone into quantity production at the Leningrad Research Institute for Vaccines and Sera.     

The use of the latex agglutination reaction for the diagnosis of a Brucella infection

The authors have developed the optimum conditions for the preparation of antigenic diagnosticum based on latex manufactured in the USSR. To sensitize latex with the diameter of microspheres equal to 0.83 microns, Brucella polysaccharide was used in a dose of 100 micrograms/ml. As stabilizer, polyvinylpyrrolidone at a concentration of 0.1% was used. The specificity and sensitivity of the diagnosticum were studied in analysis of serum samples taken from 102 healthy donors and patients with infectious diseases of nonbrucellar etiology and from 120 patients with different forms of brucellosis. The specificity of the diagnosticum was found to be 94.1% and its sensitivity, 77.5%. Comparative study of the latex agglutination test with other serological tests showed that the former test is highly effective both in acute and chronic forms of the disease. A high degree of correlation between the agglutination test, Coombs' test, the passive hemagglutination test and the latex agglutination test was established (r = 0.83, 0.72 and 0.62, respectively).     

Pseudomonas aeruginosa and Proteus antigenemia and antibody formation in mono- and mixed infections in patients with suppurative inflammatory diseases

P. aeruginosa and Proteus antigenemia and antibody production have been studied in 335 patients with purulent inflammatory diseases. The study has revealed that in the association of P. aeruginosa and Proteus with staphylococci and representatives of the family Enterobacteriaceae the level of antigenemia is considerably lower than in monoinfections or in the association of these microorganisms with streptococci. In mixed infections humoral immune response develops later than in cases of monoinfection. An important role of ecological and physiological relationships between microorganisms in the course of purulent processes and in their influence on the host has been confirmed. The use of the enzyme immunoassay in the clinical practice has made it possible to determine the etiological role of P. aeruginosa and Proteus in the development of suppurative-inflammatory diseases, to select adequate immunotherapy, including the use of anti-P. aeruginosa and anti-Proteus plasmas, and to evaluate the effectiveness of treatment.