Quantitative determination of the soluble O antigen of Salmonella serogroup B and of specific antibodies in different classes by using immunoenzyme analysis

The results of measurements of S. typhimurium O-antigen and specific IgA-, IgG- and IgM-antibodies in 115 serum samples from patients with salmonellosis induced by group B salmonellae are analyzed. As determined in this study, the concentrations of IgA-antibodies ranged 0-15 micrograms/ml, the minimal diagnostically significant value being 3.6 micrograms/ml; the concentrations of IgG-antibodies ranged 0-13 micrograms/ml, the minimal diagnostically significant value being 1.3 micrograms/ml; and the concentrations of IgM-antibodies ranged 0-50 microgram/ml, the minimal diagnostically significant value being 5.0 micrograms/ml. The minimal diagnostically significant concentration of O-antigen, determined in selected serum samples obtained from healthy donors, was 0.1 microgram/ml. There is a significant correlation between the concentrations of IgA-, IgG- and IgM-antibodies, but the concentrations of these antibodies do not correlate with the concentration of soluble O-antigen. The study showed that the simultaneous determination of S. typhimurium O-antigen and specific antibodies in the material under test significantly enhances the effectiveness of the serodiagnosis of the disease.     

The role of IgM in paratyphoid immunity

The authors analyze the results of studies on the effect produced by the immunization of calves with paratyphoid vaccine on the production of agglutinins, changes in the blood serum immunoglobulin levels, and the specificity of IgM to Salmonella dublin and Salmonella typhimurium antigens. The highest level of agglutinins to paratyphoid antigens in the blood sera of the immunized calves was registered on days 14-21 after immunization, changes in the levels of different immunoglobulin classes being insignificant. The agglutination test and the enzyme immunoassay have revealed that antibodies to S. dublin anb S. typhimurium antigens belong to IgM.     

Somatic Antigen 2 Inheritance in Salmonella Groups B and D

Somatic (O) antigen 2 of Salmonella paratyphi A replaced somatic antigen 4 of an S. typhimurium recipient as the consequence of mating with an S. paratyphi A var. durazzo Hfr strain. The genetic determinants of these O antigens behaved in this cross as alleles of a common O locus, which is linked to the determinant of histidine biosynthesis, his. By employing phage lysates obtained by growth of P22 on an S. typhimurium hybrid which had received his and O-factor 2 determinants from the S. paratyphi A Hfr, it was possible to cotransduce the his and O-antigen 2 genes to both S. typhimurium and S. typhosa. S. typhimurium transductants which received somatic antigen 2 concurrently lost O-antigen 4, and S. typhosa transductants receiving O-antigen 2, lost their native O-antigen 9. These results indicate that the genetic determinants of O-antigens 2, 4, and 9 occupy the same O locus in S. paratyphi A, S. typhimurium, and S. typhosa, respectively, and are probably allelic.     

Use of an artificial antigen simulating the Salmonella O-determinant 4 for determining antibodies to Salmonella group B O-antigen in immunoenzyme analysis

The use of the artificial antigen abequosylmannoside copolymer with acrylamide in the enzyme immunoassay for the determination of antibodies in the sera of salmonellosis patients has enhanced the specificity of the serological diagnosis of group B salmonellosis in comparison with the use of the natural antiren, S. typhimurium lipopolysaccharide.     

Determination of the O antigen of Shigella sonnei by a competitive immunoenzyme method

A technique for immunoenzymatic diagnosis of dysentery by Shigella sonnei O-antigen was developed. For induction of antibodies to O-antigen rabbits were immunized by intravenous administration of a commercial antidysentery vaccine. Specific antibodies to O-antigen belonging to class G immunoglobulins and not binding to O-antigens of Sh. flexneri and Salmonella typhimurium were obtained. beta-Lactamase of Bacillus licheniformis 749/c was used as a marker enzyme in the immunoenzymatic assay. To increase the sensitivity, beta-lactamase molecules were preliminarily linked with glutaric aldehyde into oligomers. Conjugates of Sh. sonnei O-antigen with the oligomers of B. licheniformis 749/c beta-lactamase were prepared with the periodate method by oxidizing O-antigen. The conjugate was used in competing solid phase immunoenzymatic assay for determination of Sh. sonnei O-antigen in blood serum of patients with dysentery. The sensitivity of the assay is 0.5-1 ng per 1 ml of O-antigen.     

Rapid decay of Salmonella flagella antibodies during human gastroenteritis: a follow up study

An indirect enzyme-linked immunosorbent assay (ELISA) based on Salmonella re-polymerized flagella was employed to measure levels of immunoglobulin (Ig) G, IgM and IgA antibodies in sera from 303 Danish patients diagnosed with either Salmonella enteritidis or Salmonella typhimurium. The antibody-levels were assessed at one, three and six months after onset of salmonellosis, and sera from a control-group of 170 healthy blood donors were additionally analysed in order to establish cut-off values for the analysis. Cross-reactions to other Salmonella serotypes, as well as to Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli and Helicobacter pylori were observed. At one month after onset of symptoms, 70% of the patients recovering from a S. enteritidis infection carried detectable levels of anti-flagella antibodies, as did 77% of the patients recovering from S. typhimurium infection. Three months after onset of symptoms these detection rates had decreased to 46% and 40%; and six months after onset of symptoms the detection rates were 34% and 38%. This rapid decrease in the serum levels of flagella antibodies is in conflict with the "common knowledge" statement of a long-lasting anti-flagella immunoresponse. The present study suggests that such a tenacious statement is (or may be) inaccurate.     

Effect of acetylation (O-factor 5) on the polyclonal antibody response to Salmonella typhimurium O-antigen

Antibodies directed against lipopolysaccharide (LPS) O-antigen are often critical in the immune response to Gram-negative pathogens. Mice were orally immunized with isogenic strains of Salmonella typhimurium that differ only in a minor modification of O-antigen, namely acetylation, mediated by the oafA locus. To specifically examine the effect of acetylation on the antibody response to O-antigen, antibody titers were determined against both acetylated and unacetylated LPS by ELISA. In mice immunized with an oafA+ strain, the median titer against acetylated LPS was 32-fold higher than the titer against unacetylated LPS. Mice immunized with the oafA- strain had an 8-fold higher titer against unacetylated LPS. Thus, acetylation of O-antigen alters recognition by the vast majority of individual antibodies. This differential antibody recognition of O-antigen had a statistically significant correlation with protection against subsequent challenge with virulent S. typhimurium.     

Specific anti-Yersinia G- and M-antibodies in healthy blood donors and in patients with alimentary intoxication

The work deals with the results of determination of specific antibodies in blood donors of Moscow and Tula and in patients with alimentary toxicoinfection, made with the use of enzyme immunoassay on the basis of Yersinia enterocolitica lipopolysaccharides (LPS), serovars O3 and O9. The sera of patients with alimentary toxicoinfection were found to yield positive reactions with Y. enterocolitica LPS in 35.9% of cases (the number of such reactions obtained with blood donor sera was 3 times less). The presence of cross reactions between Y. enterocolitica LPS and the microsomal antigens of the thyroid gland was established. A high detection rate of antibodies to the microsomal antigens of the thyroid gland among blood donors of Tula was registered.     

High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli.

Lipopolysaccharide from smooth strains of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli O111:B4, O55:B5, and O127:B8 was fractionated by gel filtration chromatography. All lipopolysaccharide samples separated into three major populations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from S. typhimurium and S. minnesota indicated that the three peaks were made up of molecules with average O-antigen lengths of (i) 70 or more repeat units, (ii) 30 and 20 repeats units in the samples from S. typhimurium and S. minnesota, respectively, and (iii) 1 repeat unit. In contrast to the Salmonella samples, peak 1 from the E. coli samples was not detected on polyacrylamide gels and lacked detectable phosphate. This high-molecular-weight material had a sugar composition similar to that of O-antigen and was tentatively identified as capsular polysaccharide. Peaks 2 and 3 of the E. coli samples were analogous to those of the Salmonella isolates, containing lipopolysaccharide molecules with averages of 18 and 1 O-antigen repeat units, respectively. These lipopolysaccharide molecules did not completely dissociate during electrophoresis, and multimers were detected as distinct, anomalous, slow-migrating bands. Increasing the concentration of sodium dodecyl sulfate in the gels resulted in the dissociation of these multimers.     

Assessment by immunoenzyme analysis of the immune status of donors with reference to the viruses of rubella, measles and herpes simplex

The results of testing the blood sera obtained from donors at a blood transfusion center in Moscow for the presence of antibodies to rubella, measles and herpes simplex viruses, carried out by means of the enzyme immunoassay with the use of the corresponding test systems, are presented. Antibodies to rubella, measles and herpes simplex viruses have been detected, respectively, in 81.5, 96.7 and 100% of blood sera. The proportion of sera with low, medium and high antibody titers has proved to be virtually the same with respect to antibodies to rubella and herpes simplex viruses, the sera with medium antibody titers constituting 59%. At the same time tests for measles antibodies have shown the prevalence of sera with low titers (49.2%) with the highest percentage of seronegative donors (18.5%, as compared with 3.3% in rubella and the absence of negative sera in herpes simplex).     

Use of immunoenzyme analysis in the rapid diagnosis and study of the characteristics of circulating O-antigen in biological fluids of patients with acute dysentery Sonne

The data on the clinical approval of the original enzyme immunoassay system for the determination of somatic O-antigen in the blood serum and urine of patients with acute Sonne dysentery are presented. The level of the antigen determined in the biological fluids of patients has been shown to depend on the severity of the disease. Different types of dynamic curves, reflecting the level of O-antigen in the biological fluids of patients with acute Sonne dysentery and characteristic of different clinical forms of the disease, have been established.     

Determination of antibodies against ribosomes and various cell wall components and detection of circulating antigens of group A Streptococcus in patients with erysipelas

The level of antibodies to the ribosomes, polysaccharide A and peptidoglycan of group A streptococcus in the blood of patients with primary, secondary, and often relapsing erysipelas was studied by means of the enzyme immunoassay with the use of the sandwich techniques. For control, the sera of healthy donors were used. In the sera obtained from all groups of erysipelas patients a significant rise in the levels of antibodies to ribosomes and peptidoglycan in comparison with the controls was revealed. An increase in the level of antibodies to polysaccharide A was revealed only in patients with frequently relapsing and secondary erysipelas. Depending on the clinical form and the duration of the disease, polysaccharide A was detected in 32-51.9% of erysipelas patients and protein-ribosomal antigen was detected in 28.6-51.9% of such patients.     

Systemic immunological memory in enteral immunization with the O antigen from S. sonnei

Mice received S. sonnei O-antigen at various concentrations (0.01-20,000 micrograms/ml) in drinking water. Systemic immunological memory, induced by feeding with O-antigen, was manifested by secondary immune response to parenteral boosting with homologous O-antigen or ribosomal vaccine. A pronounced priming effect was also produced by O-antigen at concentrations as low as 0.01 micrograms/ml after courses of feeding as short as 1-3 days. Even high doses of the antigen had no tolerogenic activity. The state of immunological memory was formed at least 12 days after the first feeding and lasted for a long period (at least 4 months after the last feeding). The specificity of immunological memory was proved in experiments with heterologous O-antigen (Salmonella typhimurium): the insignificant stimulating action of this antigen was revealed only when high concentrations of the antigen (1000 micrograms/ml) were used for feeding.     

Antigenic structure of Salmonella L forms studied by an immunoferritin method

The antigenic structure of the L-forms of salmonellae (S. typhimurium and S. typhi) in comparison with that of the initial bacterial cultures and revertant cultures was studied with the use of the immunoferritin method. The L-forms of salmonellae were found to retain an insignificant amount of O-antigen, as well as to have K-antigen on the surface of the cytoplasmic membrane, but in a lesser amount than the initial strains. In the cultures reverting from the L-forms of S. typhimurium and S. typhi the quantitative and qualitative characteristics of O- and K-antigens were completely restored.     

Comparative effectiveness of the bacteriologic method and of the direct determination of the O antigen of the causative agent in research on the excretions of patients with salmonellosis due to Salmonella typhimurium

The diagnostic effectiveness of the direct determination of Salmonella O-antigen in different excretions of patients with S. typhimurium infection and other acute intestinal diseases has been studied. According to the occurrence of the antigen in different kinds of excretions, they are arranged in the descending order as follows: feces, urine, saliva. The parallel serological and bacteriological study of feces enhances the number of positive results 1.6 to 2.4 times in comparison with the bacteriological study alone. The occurrence of the antigen in excretions does not depend on the age of the patients and on the severity of the disease. The indication of Salmonella O-antigen may serve as an effective supplement to bacteriological study.     

Comparative immunoenzyme analysis of sera for the presence of antibodies to a preparation of Staphylococcus aureus teichoic acids and DNA

The comparative analysis of the titers of antibodies to the preparations of S. aureus teichoic acids and DNA in the sera of healthy donors and patients with infectious endocarditis and rheumatic carditis was made by means of ELISA. The sera of patients with infectious endocarditis and rheumatic carditis, in contrast to the sera of healthy donors, showed the presence of antibodies to DNA in 23.5-76.2% of cases. The correlation between the presence of antibodies to S. aureus teichoic acids and DNA in the sera of the patients was weakly pronounced.     

Possibility of detecting human embryonal leukemia antigen]

A possibility of detecting embryonic leukemic antigen on human leukemic blast cells in an acute human leukemia cytotoxicity test with the sera and 7S and 19S serum immunoglobulins of the placental blood was studied. The presence on the blast cells of patients suffering from acute leukemia of an antigen detectable by antibodies of placental blood (of parturients) was demonstrated; this antigen was absent on the leukocytes of healthy donors.     

Role of lipopolysaccharide and complement in susceptibility of Escherichia coli and Salmonella typhimurium to non-immune serum

The role of lipopolysaccharide (LPS) in the susceptibility of Escherichia coli and Salmonella typhimurium to non-immune human serum was investigated using serum-sensitive strains of both enterobacteria. LPS from serum-resistant strains of E. coli and S. typhimurium could activate and completely remove the serum bactericidal activity, and also showed dose-dependent anti-complement activity. These properties were mainly due to the high-molecular-mass LPS: the low-molecular-mass LPS from serum-resistant strains of E. coli and S. typhimurium had only a slight effect on the serum bactericidal activity, and showed only low anti-complement activity, even at high concentration. The results suggest that LPS composition, especially the O-antigen polysaccharide chains, contributes to the susceptibility of E. coli and S. typhimurium strains to complement-mediated serum bactericidal activity.     

The mosaic of proteins synthesized by Salmonella typhimurium

A profusion of proteins with heterologous serological specificities was synthesized by S. typhimurium grown on artificial media; accordingly, sera prepared in rabbits with these proteins displayed an abundance of antibodies reacting, in agar gel, against numerous heterologous proteins. the absorption of the sera with different Enterobacterial proteins proved that the S. typhimurium proteins are a mixture of specific proteins, and common E. coli and Salmonellae determinants; in addition, a group of strongly cross-precipitating proteins common to S. typhimurium and S. choleraesuis and to S. typhimurium and S. kentucky were identified that were not present in the proteins common to S. enteritidis, S. typhi and E. coli, or in the S. paratyphi A proteins used absorptions. The specific proteins of S. typhimurium were synthesized on artificial media in, apparently, smaller amounts than the common proteins; their role in the protection of mice against infection with their natural pathogen was, however, proof of their specificity and contrasted with the ineffectiveness, in protecting the mice, of the common proteins.     

Immunoenzyme analysis of the serological activity of the structural modifications of a synthetic antigen simulating the O-determinant 4 of Salmonellae

The comparison of the copolymer of 2-O-(alpha-abequosy)-alpha-alyl-alpha-mannopyranoside and acrylamide (AMA), used as a synthetic antigen, with its natural prototype, Salmonella typhimurium lipopolysaccharide, in the enzyme immunoassay (EIA) has revealed that the serological activity of AMA is related to the specific content of antigenic determinants in its molecule. The analysis of the serological specificity of AMA indicates that this specificity corresponds to factor 4 of Salmonella O-antigen. The high activity and monofactor specificity of the synthetic antigen AMA suggest that the use of this antigen in EIA as a diagnostic preparation holds good promise.