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.     

Results of an epidemiological study of familial foci of salmonellosis in the Western Urals

The study of 449 family foci of salmonellosis demonstrated the possibility of detecting cases of salmonellosis, not diagnosed by clinical and bacteriological methods, in the passive hemagglutination test. Such undetected Salmonella carriers, not observing the rules of personal hygiene, contributed to the contamination of household articles, foodstuffs and the environment. The formation of the family foci of salmonellosis was facilitated by the belated detection and hospitalization of salmonelloses cases, as well as by the inadequate observance of the rules for infant care and feeding.     

Erythrocyte diagnostic agents for detecting bacterial antigens of the genus Citrobacter

Citrobacter antigenic and antibody erythrocyte diagnosticums, serogroups 1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, and 22, have been developed. Tests with the use of these diagnosticums have proved to be highly sensitive and mainly group-specific. The antigen in the cellular form is best detected by means of the passive hemagglutination test and in the molecular form, by means of the neutralization test. The antibody-binding and agglutinating activities of strictly group-specific and cross-reacting O-antigenic determinants differ in their sensitivity to heating and to treatment with phenol. In the study of fecal samples taken from patients the above method for the detection of Citrobacter antigens has been shown to have high resolution.     

The use of the indirect hemagglutination reaction for the serodiagnosis of HIV infection

An assay system, based on the passive hemagglutination test and permitting the serodiagnosis of HIV infection with correct results in more than 99% of cases, has been developed. Three kinds of freeze-dried erythrocyte diagnostica (with shelf life exceeding 6 months), possessing high serological activity and sensitized with recombinant gene-engineering polypeptides, have been obtained. The proposed assay system is highly promising for mass examination of sera for the presence of antibodies to HIV due to the simplicity of assay techniques, the possibility of storing the diagnostica within a wide range of temperatures (4 degrees-30 degrees C) and obtaining results in a short time (3 hours).     

Isolation and characteristics of Shigella antigens during the course of dysentery

In 2,436 fecal samples and 1,272 urinary samples taken from 633 patients with dysentery caused by S. flexneri, S. newcastle and S. sonnei, Shigella antigens were detected by means of the passive hemagglutination test with antibody diagnostic agents and the antibody neutralization test. The antigen-binding activity of shigellae in urine dynamically increased in the course of dysentery. The comparison of the parallel results of both serological tests made it possible to evaluate the dispersion of the released antigen: it dynamically increased in the course of the disease, this increase being particularly high in feces. The dispersion of Shigella antigens in urine was greater than in feces over the entire course of the disease. These regularities in the release of the antigens and especially the specific features of the serological tests determined the scheme of the serological diagnosis of dysentery by the indication of Shigella antigens.     

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.     

Antigenic characteristics of standard strains of Providencia rettgeri (O- and H-antigens)

Additions and changes have been introduced into the existing antigenic diagnostic scheme of P. rettgeri on the basis of the study of the antigenic structure of standard strains from foreign collections: new, formerly unknown varieties of somatic and flagellar antigens (O35, O36, H27, H28) have been discovered, the complex of antigenic factors for H-antigens 7, 10, 23, 27 has been discovered. Strains 958 (36 : 28) and 979 (16 : 27a, 23b, 2a), previously classified with the genus Morganella, have been identified by O- and H-antigens.     

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 possibility of using the demonstration of Shigella antigens in the study of the epidemic process in dysentery

The detection of Shigella antigens in feces in accordance with the results of the passive hemagglutination test is more effective than the bacteriological method for finding out persons infected with Shigella, especially in the absence of pronounced clinical manifestations of dysentery, and can be used in the study of the mechanism of the epidemic process. The examination of two groups, each consisting of permanent and temporary members, has revealed that the use of this method for detection of the antigen makes it possible to perform the epidemiological analysis of morbidity and, in particular, to determine the source(s) of infection.     

The development of methods for obtaining monospecific ingredients for immunoenzyme analysis

The methods of the modification of Salmonella O- and H-antigens and the preparation of biologically active sorbents on their basis have been developed. The use of these sorbents has permitted the isolation of affinity antibodies with strictly defined specific activity. The work shows the possibility of the successful use of carriers obtained on the basis of porous glass, chemically modified by acrylic copolymers containing activated carboxylic groups, and intended for the immobilization of antigens of both protein and carbohydrate nature.     

Use of the toxin-tissue receptor reaction for detecting toxic substances of the causative agents of acute intestinal diseases

The possibility of using erythrocytic ganglioside diagnostic reagents (EGDR) for the detection of V. cholerae, E. coli and S. typhimurium enterotoxins in the passive hemagglutination (PHA) test has been shown. Museum strains and cultures isolated from patients with acute intestinal diseases were tested for the presence of enterotoxins. Cell-free extracts were studied by biological methods and by serological titration in the PHA test with the use of EGDR. The diagnostic reagent was found to interact only with those enterotoxins whose specific receptors were gangliosides GM1.     

Specificity of the serological diagnosis of dysentery using erythrocytic reagents

The serological specificity of the diagnosis of dysentery, made by different methods with the use of antigenic and antibody erythrocyte diagnostic agents under the conditions of the circulation of different Shigella species and subspecies in a given locality, has been compared. The method for the determination of the diagnostic titer of the total serum antibody activity has proved to be the least specific. The methods for the detection of Shigella antigens, especially over time, in patients' excretions and for the determination of serum antibody activity show complete specificity. The combination of specificity and sensitivity makes the detection of Shigella antigens in feces by means of the passive hemagglutination test and the antibody neutralization test the method of choice for the diagnosis of dysentery.     

Heterogeneity of antibody response to Salmonella lipopolysaccharide measured by passive hemagglutination and hemolysis in mice

The complement-requiring passive hemolysis test with Salmonella typhimurium lipopolysaccharide-coated sheep erythrocytes is more sensitive for antibodies directed against the lipopolysaccharide than is the passive hemagglutination test. The hemagglutinating and hemolyzing antibodies produced in Swiss mice by hyperimmunization, either with or without Freund's adjuvant, were distributed in both the light and heavy fractions isolated by sucrose density gradient fractionation and gel filtration. IgM fractions, whether tested by hemagglutination or hemolysis, were sensitive to 2-mercaptoethanol (0.15 m). On the other hand, IgG hemolytic antibodies were more sensitive to 2-mercaptoethanol than were IgG hemagglutinating antibodies. The resistance of IgG hemagglutinating activity amounted to about 72 to 95% of the total IgG recovered, whereas the resistant portion of the IgG hemolytic activity was approximately 40 to 53%. It is suggested that, although mercaptoethanol sensitivity is not a definitive test for IgM antibody, its use in connection with the hemagglutination test gives at least an approximation of the IgG antibody, whereas the hemolysis test gives a better approximation of maximal measurable antibody against Salmonella lipopolysaccharides.     

The fibronectin binding of salmonellae studied by a passive hemagglutination method]

The study has shown that the fibronectin-binding properties of different Salmonella species can be tested by means of passive hemagglutination with fibronectin-sensitized formulated sheep red blood cells (SRBC). S. enteritidis and S. typhimurium isolated from foodstuffs, as well as the representatives of the species S. kottbus, S. anatum, S. tshiongwe and S. newlands, are capable of binding fibronectin. Fibronectin-binding properties have also been detected in Shigella sonnei, as well as in eubiotic microorganisms Escherichia coli M 17 and Lactobacillus plantarum. In pH ranging from 6.5 to 7.4 salmonellae, shigellae and E. coli agglutinate fibronectin-sensitized SRBC much more effectively in acidic pH values, while changes in pH have been found to produce no essential influence on the fibronectin-binding capacity of Staphylococcus aureus and lactobacilli.     

An evaluation of the scope of the circulation of Salmonella among the workers of commercial poultry- and meat-packing enterprises based on serological study data in the PHA test]

Serum samples from 641 workers of large poultry and meat-packing plants were studied in the passive hemagglutination test with the use of Salmonella complex and serogroup diagnostica. A specific increase in the level of anti-Salmonella antibodies in 60.7% of poultry plant workers and in 9.8% of meat-packing plant workers was established. Among the workers of the poultry plants the most pronounced immune shifts were detected in persons having contacts with sick poultry and pathological material and among the employees of the meat-packing plant, in those who ate raw sausage meat. A high level of antibodies in the professional groups under study was observed as early as in the first year of work at the plant and persisted over the whole period of this work. Under the conditions of constant contamination of the workers of poultry and meat-packing plants with small doses of salmonellae specific immunity to this infection was seemingly induced, which inhibited the development of the manifest forms of infection, but did not prevent the formation of chronic carrier state.     

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.     

Accelerated laboratory diagnosis of bacterial pneumonia

The effectiveness of countercurrent immunoelectrophoresis (CIE) used for the accelerated differential diagnosis of pneumococcal, staphylococcal, mycoplasmal and Legionella infections in cases of pneumonia has been shown. The presence of correlation between the results obtained in the bacterial study of sputa and bronchial washings and in CIE has been revealed, which gives grounds for recommending CIE for the accelerated diagnosis of pneumococcal pneumonia on the basis of the analysis of sputa, bronchial washings and blood sera obtained from patients. In Legionella infection the passive hemagglutination test with antigenic diagnostica has proved to be more effective for accelerated diagnosis than CIE.     

Use of latex test for detection of Salmonella in meat]

The aim of this study was to examine the usefulness of latex test for detection of Salmonella in raw ground meat . Five hundred and fifty samples of meat were examined, including 368 samples artificially contaminated with S. enteritidis and S. typhimurium. Samples for latex test were also derived from classical microbiological culture (2 ml) which was run in parallel. Coincidence of positive results obtained in latex test with positive results in microbiological method was 7.6% to 15.3% (after introductory multiplication) and from 38.2% to 73.9% (after selective multiplication). There was no bacteriological confirmation for 7 samples (3.9%) positive in latex test. Ground meat environment and its bacterial flora have no qualitative influence on a result of latex test; the detection of Salmonella takes place if there is a sufficient concentration of somatic antigens of these microorganisms in a tested sample. However, these factors as well as a method od preparation of bacterial culture have influence on the sensitivity of latex test. In the light of this study it seems possible to use latex test for selection of samples. Positive samples could be eliminated from further bacteriological examination. The further studies are necessary involving other types of food products and studies on optimalisation of preparation of samples for latex test are also required.     

A trial application of the latex test for evaluation of antibody level in rabbit immune sera]

A latex test was elaborated which served for evaluation of quality of rabbit immune sera for antigen 0 of selected Gram-negative bacteria. Sensitivity and specificity of this test in comparison with passive hemagglutination and immunoenzymatic DOT-ELISA reactions was evaluated. These studies were performed on immune sera for antigen O of Salmonella groups B, C1, C2, D and E, Yersinia pseudotuberculosis and in antigen preparations for above listed microorganisms both in homologous and heterologous systems. It was found that sensitivity of the latex test is 9 to 160 times lower than that of passive hemagglutination and 7 to 307 lower than for DOT-ELISA. Sensitivity of the latex test and passive hemagglutination reaction was evaluated on the basis of results of cross reaction between studied antigens and unabsorbed rabbit sera, establishing so called sensitivity indexes, which were informing how many times heterologous titer is lower than homologous titer. So evaluated sensitivity of the latex test was close to sensitivity of the passive hemagglutination reaction. It was found that slide latex test is characterized by satisfactory sensitivity and good sensitiveness and may be used for evaluation of antibody level 0 antigens of Salmonella and Yersinia. The value of this test is characterized by high repeatability of results, as well as low work and time-consuming.     

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.