Homologous Immune Serum Research Articles

Immunochemical activity of <i>Yersinia pseudotuberculosis</i> ompF and ompC porins evaluated by optical trapping

Introduction. Study of features for interacting antigen-antibody system is of great importance for developing new modern tools for diagnostics and therapy of infectious diseases. In this regard, it is of great interest to study the rupture force between bacterial antigens and antibodies using modern biophysical methods including optical trapping. The importance of surface antigens in the immunochemical activity of Yersinia pseudotuberculosis assessed by such method has not been evaluated yet. In this work we examined an opportunity to evaluate the interaction of hydrophobic Y. pseudotuberculosis porins OmpF and OmpC with specific antibodies using optical trapping method. Materials and methods. Polystyrene microspheres (d = 1 m) were coated by passive adhesion with purified preparations of OmpF and OmpC porins; microsphere sensitization was verified by enzyme immunoassay. Antibodies from mouse sera were adsorbed onto the glass surface by chemical linking. The rupture force in the porins-antibodies system was determined using a laser trap according to the previously developed algorithm. Results. Using a model system including polystyrene microspheres sensitized with the proteins and aminated glass substrate coated with immune or nonimmune serum, significant differences in binding strength of OmpF and OmpC porins to homologous immune versus nonimmune sera were detected. The average forces of interaction with immune sera was 60 pN for OmpF microspheres (control 40 pN) and 69 pN for OmpC microspheres (control 49 pN). The proportion of irreversible substrate binding of the microspheres coated by the antigens to the treated with immune vs. non-immune sera was significantly higher. The results of assessing the average interaction force, as well as the predominance of the proportion of irreversible binding of antigen-coated microspheres with sera-treated substrates, indicates that specific interactions contribute significantly to the force of interaction. The aforementioned method can be used to evaluate the forces of intermolecular interaction in similar model systems using other microbial antigens.

Efficacy of glutathione-S-transferase purified antigen of the gastro-intestinal nematode Haemonchus contortus in diagnosis of sheep haemonchosis.

Haemonchus contortus is one of the most pathogenic and economically important parasites of sheep. Different H. contortus antigens; crude somatic antigen (CSA), excretory/secretory antigen (ESA), crude larval antigen (CLA), glutathione-S-transferase antigen (GST) and recombinant protein (rhcp 26/23) were prepared and characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The antigens were immunologically evaluated through indirect enzyme linked immunosorbent assay (ELISA) for diagnosis of haemonchosis in experimentally and naturally infected sheep. Analysis of the resultant bands of SDS-PAGE demonstrated that 13, 6, 11, 2 and 1 protein bands from CSA, ESA, CLA, GST and rhcp 26/23, respectively and analysis of the resultant bands of western blot showed that 13, 6, 4 and 1 reactive bands detected from CSA, ESA, CLA and GST, respectively. The results of ELISA of different antigens revealed that sero-prevelance of CSA, ESA, CLA, GST and rhcp 26/23 were 78.51, 82.34, 85.319, 45.319 and 90.8% respectively, sensitivity were 100, 90, 100, 96.66 and 90%, respectively and specificity were 0, 70, 10, 70 and 6.66%, respectively with diagnostic potency were 50, 80, 55, 83.33 and 48.33%, respectively. Statistical analysis using Chi square test found that GST is the best one that can be used. The cross reactivity of GST antigen, crude Fasciola antigen and crude Moniezia antigen tested versus their homologous hyper immune sera at different dilutions using ELISA. The current study reported that GST antigen could be considered as a promising antigen for diagnosis of haemonchosis.

Phagocytosis of Streptococcus uberis by bovine mammary macrophages: opsonizing effect of bovine antiserum.

Five encapsulated and five non-encapsulated strains of Streptococcus uberis were tested for their ability to resist phagocytosis and intracellular killing by bovine mammary macrophages obtained during the non-lactating period. The percentage of phagocytosis and intracellular killing of encapsulated strains ranged from 43 to 51 and from 25 to 40, respectively. By contrast, the percentage of phagocytosis and intracellular killing of non-encapsulated strains ranged from 66 to 74 and from 65 to 75, respectively. Two encapsulated and two non-encapsulated strains were opsonized with homologous and heterologous pre-immune and immune bovine sera. Phagocytosis and intracellular killing of encapsulated strains increased significantly when opsonized with serum of cows vaccinated with a homologous and heterologous encapsulated strain. By contrast, no significant differences were observed when serum from cows vaccinated with a non-encapsulated strain was used. These results suggest that resistance to phagocytosis is a common feature among encapsulated strains of S. uberis, and that cross-reactivity exists between encapsulated strains.

Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains.

The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco-Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya-Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elution. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite-encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surface-specific immunoprecipitation reaction of 35S-methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite-encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.

Specificity of the carbon immunoassay (CIA) test for the diagnosis of Toxoplasma infections

A rapid and low cost procedure, the carbon immunoassay (CIA) test, was evaluated for the diagnosis of Toxoplasma gondii infections. Using a closely related parasite ( Besnoitia jellisoni) as antigen, and homologous or heterologous immune sera, it was demonstrated by light and electron microscopy that CIA is a very reliable and specific test. As it is neither expensive nor time-consuming, it can be recommended for general and routine laboratory use.

Selection of aBorrelia burgdorferiantigenic variant by cultivation in the presence of increasing amounts of homologous immune serum

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.

Selection of a Borrelia burgdorferi antigenic variant by cultivation in the presence of increasing amounts of homologous immune serum

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.

Haemophilus ducreyi, a cytotoxin-producing bacterium.

An extra- and an intracellular product from the bacterium Haemophilus ducreyi were shown to have a cytotoxic effect on cell lines of epithelial origin, e.g., HEp-2 and HeLa cells. The cytotoxic effect appeared on cells within 24 h and resulted in cell death and morphologically changed cells. The cytotoxic activity was heat and pronase sensitive. The activity could be removed by incubation with the target cells, suggesting attachment of the agent to the cells. The cytotoxic products were secreted into the environment during exponential growth of bacteria and produced by most of the H. ducreyi strains tested. The activity was neutralized by homologous rabbit immune serum but not by the corresponding preimmune serum. The results indicate that strains of H. ducreyi produce a cytotoxin which may be of importance to the pathogenesis of chancroid.

Clonal and temperature induced differences in serotypes in the ciliate Dileptus anser

Antigenic differences have been demonstrated in 20 clones of the ciliate Dileptus anser by immobilization assay, precipitation reaction in agar and immunofluorescence, using antisera obtained against two of these clones. Homologous clones reacted with antisera in high dilutions. 7 heterologous clones demonstrated a slight reaction of immobilization. 13 heterologous clones failed to react with immune sera, which allowed to attribute them to some other serotypes. By immunodiffusion methods the presence of an antigen expressed in a single clone has been shown. When testing a certain clone with the homologous immune serum by immobilization reaction and immunofluorescence, the subclones cultured at 17°C and 25°C express two different serotypes each. When cultivating the same clone at 19 °C, 21 °C or 23 °C, the cells express both of these serotypes, although to a different extent depending on the temperature of their cultivation. Heterologous clones often show cross-reactions with the same immune sera, usually when the ciliates are cultivated at the same temperature as the cells used for immunization. The change of cultivation temperature from 17°C to 25 °C, or vice versa, leads to a gradual replacement of one serotype with another. This transformation of serotypes induced by the change in cultivation temperature was completely reversible, and judging by the immunofluorescence data, occurred in most of, if not in all, the cells. The new serotype appeared and the old one disappeared at the same rate and took 6 to 7 days at 17 °C and 3 to 4 days at 25 °C. In both cases 6 to 8 cell divisions took place during that time.

Structural and antigenic analysis of Chlamydia pneumoniae

Several isolates of Chlamydia pneumoniae were compared with each other and to Chlamydia trachomatis and Chlamydia psittaci by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein profiles of the C. pneumoniae isolates appeared to be the same and were distinct from the other Chlamydia species. A 39.5-kilodalton (kDa) protein, similar in molecular weight to the major outer membrane proteins (MOMP) of C. trachomatis and C. psittaci, was found in the Sarkosyl-insoluble fraction, demonstrating its association with the outer membrane complex. In the outer membrane complex, the MOMP was shown to exist in disulfide-linked protein complexes. Electron microscopy of the Sarkosyl-extracted elementary bodies showed that the structural rigidity and pear-shaped morphology remained intact. Rabbit immune sera prepared against C. pneumoniae demonstrated immunoreactive proteins of 98-, 77-, 75-, 66-, 60-, 39.5-, 28-, and 17.5-kDa proteins. Cross-reactivity experiments revealed that most of the antigenic reactivities shared between C. psittaci and C. trachomatis extend to C. pneumoniae and that the 98-kDa protein recognition appeared to be C. pneumoniae specific. In contrast to the other Chlamydia spp., the recognition of the C. pneumoniae MOMP by homologous immune sera was weak and was cross-reactive with the MOMPs of the other Chlamydia species. These results suggest that the C. pneumoniae MOMP is less immunogenic and antigenically complex than are the MOMPs of C. trachomatis and C. psittaci.

モルモット補体を利用したＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの代謝阻止抗体測定法

Improvement of the fermentation-inhibition (FI) test for Mycoplasma pneumoniae was attempted. The sensitivity of detecting the FI antibody to M. pneumoniae in the homologous immune rabbit serum was notably elevated, when such FI medium containing gamma globulin-free horse serum and guinea-pig complement in substitution for unheated horse serum was used. The M. pneumoniae suspension was filtered to remove aggregates of the organisms and used as the antigen for this new FI test. In detection of the serum antibody of patients with M. pneumoniae infections, the new FI test showed much higher sensitivity than conventional FI test and strongly correlated (r = 0.84) with high density particle agglutination test known to detect both IgG and IgM antibodies.

The effect of C3 depletion on the clearance of Trypanosoma cruzi induced by IgG antibodies

Passive transfer of homologous immune serum or IgG anti- Trypanosoma cruzi antibodies to normal mice containing circulating T. cruzi bloodstream trypomastigotes (Btrys) induces a very fast clearance of the parasites. Previous work from this laboratory has shown that F(ab′)2 fragments obtained from IgG anti- T. cruzi antibodies retain the ability to induce lysis of the Btrys but are unable to induce clearance of the parasites. This suggests that the clearance is dependent on the Fc region. The removal of the Btrys may then be effected by attachment of the antibodies to the parasites and removal of the opsonized parasites by the mononuclear phagocytic system. Attachment of the opsonized parasites to macrophages may be effected either through the Fc receptor that binds specifically to the heavy chain of IgG or through C3b fragments after cleavage of C3 by C3-convertases. In order to find out the possible role of C3b in this phenomenon the clearance of Btrys was determined in mice depleted of C3 by previous treatment with cobra venom factor. The results of these experiments showed that depletion of C3 completely abolished the immune clearance induced by anti- T. cruzi antibodies. It is suggested that C3 is required for the clearance of Btrys from circulation.

Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon.

Virus particles isolated from hatchery reared fish with infectious pancreatic necrosis (IPN) were neutralized by homologous immune sera but not by immune sera raised against IPN virus serotype 1, 2, and 3. This virus isolate, called the N1 strain, was detected in one year old Atlantic salmon (Salmo salar) during an outbreak with histopathological lesions of IPN and slightly increased mortality. The polypeptide pattern of N1 virus differed markedly from that of the three classical IPN virus serotypes. Double stranded RNA isolated from the N1 virus particles, co-migrated during agarose gel electrophoresis with nucleic acid isolated from the IPN virus Jasper and Ab strains. Nucleic acid hybridizations using low stringency washing conditions and a synthetic DNA oligonucleotide probe (representing the 3' end of the A segment of the Jasper strain) gave positive results with the IPN virus Jasper, Ab, Sp, and N1 strains. The results presented in this paper show that the N1 isolate differs immunologically and biochemically from the IPN virus serotypes 1, 2, and 3 and may represent a new serotype of IPNV.

Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi.

Humans and mice chronically infected with Trypanosoma cruzi present a strong humoral immune response mediated by specific antibodies. Passive transfer of homologous immune serum to normal mice containing circulating bloodstream trypomastigotes (Bts) induces a very fast clearance of the parasites. In order to find out the role of the different immunoglobulin classes in the clearance, mice containing a known number of these parasite forms in circulation were injected with total immune serum, IgG-free serum, IgG1, or IgG2 fractions and the speed of removal of the parasites from circulation was determined. The results of these experiments suggest that the immune clearance of T. cruzi is due to antibodies located in the IgG isotype, particularly in the IgG2 subclass.

Conservation and diversity of Campylobacter pyloridis major antigens.

Infection with Campylobacter pyloridis has been strongly associated with gastritis in humans although its etiologic significance is currently undefined. We examined the structure and antigenicity of whole-cell, outer-membrane, acid-extractable surface protein, and proteinase K-treated whole cell lysate preparations from eight C. pyloridis strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with homologous and heterologous immune rabbit serum. Whole-cell and outer-membrane profiles observed in all strains of C. pyloridis were nearly identical; none were similar to those of C. jejuni and C. fetus. Major whole-cell bands migrated at 26,000, 29,000, 56,000, and 62,000 molecular weights. The acid-extracted protein profiles of all C. pyloridis strains also were similar to one another and showed similarities with acid-extracted proteins from C. jejuni, with major bands migrating at 29,000, 48,000 to 53,000, and 62,000. All proteinase K-treated lysates showed different lipopolysaccharide (LPS) profiles, ranging from rough to smooth with multiple repeating side chains. Immunoblots of whole-cell and proteinase K-treated preparations of the C. pyloridis strains showed that there was antigenic cross-reactivity of proteins migrating at 62,000 and 56,000, but not in other regions, and cross-reactivity between LPS core regions but not side chains. These results suggest that C. pyloridis has both protein and core LPS group antigens and strain-specific protein and LPS side chain antigens.

Protection of Junín virus-infected marmosets by passive administration of immune serum: association with late neurologic signs.

Argentine hemorrhagic fever (Junín virus) is a human viral disease for which immune therapy proves effective, though a late neurologic syndrome is occasionally associated with the treatment. We attempted to determine in the infected marmoset Callithrix jacchus whether immune therapy leads to protection and/or CNS damage. Fifteen C jacchus were inoculated with 10(3) tissue culture infectious dose 50% (TCID50) of the XJ strain of Junín virus. On day 6 post infection (pi), 12 primates were treated with homologous immune serum. Animals were observed daily; and hematologic, serologic, virologic, and histologic studies were performed. All primates, both treated and controls, presented leukopenia, thrombocytopenia, anemia, and weight loss from day 14 pi onward. The three control animals died on days 22, 25, and 32 pi. Among the 12 treated monkeys, 3 died on days 21, 22, and 29. Hematologic values returned to normal during the second month; initial weight was recovered by the fourth month. Three out of the nine survivors showed neurologic alterations of various degrees, with hind-limb paralysis in the most severe case. Among treated monkeys, viremia and viral titers in the lungs, kidney, and lymph nodes were lower than in controls. Neutralizing antibodies were present in high titers in all treated marmosets, except in the one presenting paralysis in which values were minimal and viral persistence was detected in CNS. In conclusion, immune serum treatment of Junín virus-infected marmosets was found to reduce mortality from 100% to 25%. Viremia and viral titers in organs were lowered, and late neurologic signs appeared in 30% of treated survivors.

Restriction endonuclease DNA analysis of Leptospira interrogans serovars icterohaemorrhagiae and hebdomadis.

Antigenic variants of Leptospira interrogans serovars copenhageni and hebdomadis were examined by bacterial restriction endonuclease DNA analysis with EcoRI, XhoI, SalI, BstEII, and HindIII as the digesting enzymes. The antigenic variants were stable cloned strains which had been cultivated in media containing homologous immune serum. One of the strains examined has been reported elsewhere (R. Yanagawa and J. Takashima, Infect. Immun. 10:1439-1442) as having an antigenic makeup which more closely resembles serovar kremastos than the serovar hebdomadis parent. The closely antigenically related but naturally occurring serovars icterhaemorrhagiae strain RGA and copenhageni strain M20 were examined in parallel. No differences could be shown between the hebdomadis parent and any of its mutants. Serovars copenhageni and icterohaemorrhagiae produced patterns which differed in the high-molecular-weight bands only. The Shibaura parent strain did not differ from copenhageni M20, but the Shibaura M1 strain differed from the other mutants and from icterohaemorrhagiae RGA in its high-molecular-weight bands.

Campylobacter jejuni outer membrane proteins are antigenic for humans.

All Campylobacter jejuni strains have a major outer membrane protein (OMP) that migrates between a molecular weight of 41,000 (41K) and 45K and represents more than 50% of protein present, plus several more minor bands. Using 125I-radiolabeled C. jejuni cells in a radioimmunoprecipitation procedure to assess whether the OMPs were antigenic, we studied serum from rabbits immunized with C. jejuni cells, from humans convalescent after C. jejuni infection, and from appropriate controls. In this assay, the major OMP was the major antigen for both homologously and heterologously immunized rabbits and infected humans but not for controls. Minor bands at 29K and 50K were also antigenic. We tested human and animal sera in a Western blot procedure using anti-immunoglobulin A (IgA), anti-IgG, or anti-IgM conjugates. Homologous and heterologous immune rabbit serum, but not control serum, recognized a large number of membrane proteins between 15K and 91K, including the major OMP. Both Campylobacter spp.-infected and healthy humans showed IgA, IgG, and IgM responses to the major OMP, although the response was more pronounced in the former group. Sera from infected humans recognized several minor bands to a significantly greater extent than control sera did. Our data suggest that there is antigenic similarity between the OMPs of different C. jejuni strains and that some of these OMPs recognized by infected animals and humans have vaccinogenic potential.

Reactivity of human coronavirus OC43 and neonatal calf diarrhoea coronavirus membrane-associated antigens.

Human embryonic lung fibroblast cultures and Vero cell cultures infected with cell culture-adapted strains of human coronavirus (HCV) OC43 or neonatal calf diarrhoea coronavirus (NCDCV) were shown to possess highly cross-reactive membrane-associated antigens (MAA) by the indirect fluorescent antibody technique (IFAMA). MAA appeared 3 h post-infection, concurrently with the appearance of cytoplasmic antigens. Electron microscopic observations of cell cultures infected with either coronavirus strain and labelled with the immunoperoxidase antibody (IPA) technique for MAA detection showed that MAA consisted mainly of a strongly labelled, discontinuous, brush-like layer of amorphous material, strictly associated with the infected cell membrane. By light microscopy, reactivity of MAA with homologous and heterologous immune serum was similar to that of antigens detected by IPA in ethanol-fixed infected cells. IPA and IFAMA, but not haemagglutination-inhibiting (HI) and neutralizing (Nt) antibody, were strongly decreased by absorption of immune sera with trypsin-treated glutaraldehyde-fixed cell cultures infected with homologous virus. MAA IgG antibodies were detected by IFAMA in both human and animal sera. Sera from infants showing an HI and Nt, but not an IPA, antibody response to HCV OC43 were also free of detectable IFAMA antibody to HCV OC43.

Dual role of the immune response in street rabiesvirus infection of mice.

The cellular and humoral immune responses of mice to footpad injection of salivary gland suspensions of street rabiesvirus were investigated. Suppression of these responses with cyclophosphamide both increased the overall mortality rate from (50 to 100%) and delayed onset of disease signs and death for 1 to 2 weeks. Despite the absence of disease signs in these immunosuppressed animals, virus was present in the central nervous system, as shown by fluorescent-antibody staining of corneal epithelium. The onset of paralysis after limited immunosuppression was temporally related to a return to immune responsiveness, and passive transfer of homologous immune serum to infected immunosuppressed animals brought about their early paralysis and death. These findings indicate the importance of the immune response in the pathogenesis of street rabiesvirus infection.