Generation of antibodies to Yersinia pseudotuberculosis antigens using the colloid gold particles as an adjuvant

Serum titer achieved while producing antibodies to Y. pseudotuberculosis surface antigens with the use of colloid gold as adjuvant was as high as that achieved with the use of Freund's complete adjuvant (1:10,240). Still the amount of the antigen introduced when colloid gold particles were used as adjuvant was lower by 2 orders. The study revealed that colloid gold used as antigen carrier activated the phagocytic activity of lymphoid cells.     

Heterogenetic antigens of Yersinia pseudotuberculosis in human erythrocytes and organs

The study of the antigenic relationship between Y. pseudotuberculosis in red blood cells and different highly vulnerable tissues of the human body (in the liver, the spleen, the kidneys, the lymph nodes, the intestine) has been carried out; as a result, the presence of heterogeneous substances has been shown. Y. pseudotuberculosis heterogeneous antigens play an important role in the formation of severe forms of the disease and determine the frequency of relapses; they also determine the specific action of diagnostic and therapeutic preparations.     

Study of Yersinia pseudotuberculosis surface antigen epitopes using monoclonal antibodies

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O-side chains. The concentration of these epitopes increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.     

Monoclonal antibodies against tumor associated antigens

By the use of xeno-immunization, syngeneic immunization, or human lymphocytes, we prepared monoclonal antibodies against human epithelial tumors, especially against bladder tumors. Here, we describe results of antigen analyses and application to the tumor diagnosis.     

Plasmids of Yersinia pseudotuberculosis

Plasmids with the sizes of 5.7; 51; 70-77; and 120-130 kb were found in six strains among the ten strains collection of Yersinia pseudotuberculosis. The restriction endonucleases analysis. Southern-blot hybridization and physical maps construction were performed for the plasmids. The 70-77 kb plasmids were found to be analogous to the Ca2(+)-dependence plasmid pYVO19 from Yersinia pestis EV76. The difference between the plasmids of this type is in the insertions or deletions located on the similar fragments of the restriction maps. The 51 kb plasmid has no common fragments with the Ca2(+)-dependence plasmids and does not code for virulence properties of the strain harbouring it. No homology is shared by the 5.7 kb plasmid and the 10 kb plasmid from Yersinia pestis EV76. Replicon of the 5.7 kb plasmid has been used to construct the pVS11 vector plasmid.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia Yop proteins by Western-blot

The results obtained with the use of the western-blotting showed that antibodies for released proteins YopD (33-36 kDa) were the most frequently detected antibodies in serum samples from patients suspected for yersiniosis. Reactions between serum samples studied and the YopD protein were very intense, suggesting that protein is the strongest immunogen among the utilised, released proteins Yop of Yersinia. Antibodies IgM were more often diagnosed in patients with abdominal pain in the contrary to antibodies IgA which were characteristic to patients with reactive arthritis. Detailed analysis of the results of western-blotting on serum samples obtained several times from individuals with yersinosis during the course of infection in this investigation have showed also that antibodies of the IgA class hold longer in serum of individuals with arthritis compared with individuals with yersinosis not complicated by arthritis. In joint-fluid samples obtained from patients with arthritis antibodies for particular released proteins Yop were detected in the same class of immunoglobulins like in serum samples obtained from those individuals.     

A Yersinia pseudotuberculosis protein which cross-reacts with HLA-B27

The most-debated question in the investigation of the spondyloarthropathies has been whether there is molecular mimicry between host HLA-B27 antigens and the arthritis-causing pathogens. We have generated a monoclonal anti-HLA-B27 antibody in our laboratory and have used a radioimmunoassay to screen a panel of bacterial species. Two strains of Yersinia pseudotuberculosis were found to be highly reactive. The cross-reactive Yersinia component was identified by Western blot to be a 19,000 component. A preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography apparatus was constructed to isolate milligram quantities of this component. To verify that the component carried the HLA-B27-specific epitope, rabbits were hyperimmunized with the purified materials. Affinity-purified antibodies from one of the immunized rabbits indeed carried anti-HLA-B27 activity. Last, antibodies generated against synthetic peptides derived from the HLA-B27.1 amino acid sequence were tested against the Yersinia component. Positive reactivity was found with antibodies generated against a peptide spanning residues 69-83 of the HLA-B27.1 protein. Since this resides in the segment responsible for the allotypic specificity of the antigen, these experiments establish the presence of molecular mimicry to a high degree of confidence.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

Monoclonal antibodies against developmentally regulated corneal antigens

The chick cornea is comprised of three cellular layers, each associated with a discrete extracellular matrix. The absence of specific markers for these cellular and acellular components has made it difficult to investigate the cell-cell and cell-matrix interactions which occur during development of this organ. We have approached this problem by producing monoclonal antibodies to species-specific, developmentally regulated antigens of the chick cornea. By immunofluorescence staining patterns the antibodies fall into three distinct groups. One group is directed against the corneal extracellular matrix. At 9 days of embryonic development staining by these antibodies is detected at the endothelial surface (in Descemet's membrane), and in the posterior part of the stroma. During development it progresses anteriorly throughout the entire width of the corneal stroma and Bowman's membrane until, by 14 days, it is found in all three specialized extracellular matrices of the cornea. Throughout most of development these antibodies do not recognize any other ocular or nonocular tissue examined. Late in development they begin to lightly stain nerve bundles. A second group of antibodies is highly selective for the corneal epithelial cell layer. These begin to stain at 12 to 13 days of development and cause very bright fluorescence by 14 days. A third group stains the extracellular matrix of the cornea in a manner spatially and temporally identical to that of the first group, but in addition recognizes certain basement membranes. The possible relationship of the antigens recognized by these groups of antibodies to developmental events occurring at the time of their appearance, and the potential use of all three antibody groups in studying corneal development are discussed.     

Monoclonal antibodies against rat kidney antigens.

The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of special methods. With the aim of producing selective markers for these three portions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibodies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some antibodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kidney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clusters of the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.