vi—PHA试剂的研制及其在检测伤寒带者中的应用

Purified S. typhi Vi antigen is sensitized with equal volume of tannic acid treated formalational sheep erythrocytes (SRBC) at a final concentration of 1 microgram/ml. The Vi-passive hemagglutination assay (Vi-PHA) diagnostic reagent is developed to detect Vi antibodies to S. typhi for the detection of chronic carriers after typhoid fever and the screening S. typhi healthy carriers from food-handlers, which is characterized with high sensitivity, strong specificity and good stability. This Vi-PHA reagent is able to detect 1.16 micrograms/ml of Vi antibodies and doesn't make any cross reaction with healthy sera. For the sera of other diseases, the cross rate is only 0.84%. Using this reagent, 19 positive sera (6.93%) are detected from 274 convalescent sera from typhoid fever, 14 convalescents of which are stool-culture S. typhi positive, that persists a positive rate of 73.68%; 3 positive sera are detected from 106 foodhandlers, one of which is stool-culture S. typhi positive. Therefore, the reagent is simple, convenient, rapid and easy to be applicated in basic unit.     

Salmonella Typhi sense host neuroendocrine stress hormones and release the toxin haemolysin E

Salmonella enterica serovar Typhi (S. typhi) causes typhoid fever. We show that exposure of S. typhi to neuroendocrine stress hormones results in haemolysis, which is associated with the release of haemolysin E in membrane vesicles. This effect is attributed to increased expression of the small RNA micA and RNA chaperone Hfq, with concomitant downregulation of outer membrane protein A. Deletion of micA or the two-component signal-transduction system, CpxAR, abolishes the phenotype. The hormone response is inhibited by the β-blocker propranolol. We provide mechanistic insights into the basis of neuroendocrine hormone-mediated haemolysis by S. typhi, increasing our understanding of inter-kingdom signalling.     

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium

Abstract The monoclonal antibodies to porin, an outer membrane protein isolated from Salmonella typhimurium and sandwich enzyme linked immunosorbent assay (ELISA) has made possible the detection of porin from sera of S. typhimurium -infected mice. The specificity of the monoclonal antibodies was ascertained based on their cross-reactivity with porins isolated from S. typhi, Shigella flexneri and Escherichia coli and lipopolysaccharide (LPS) of S. typhimurium and E. coli . Serum samples were found to be positive for porin as early as 3 days after intravenous and 5 days after oral infection. In addition, a positive correlation was observed between the bacterial load and the concentration of porin detected in the sera. On the other hand, analysis of sera for anti-porin antibody showed diametrically opposite time kinetics with antigenaemia. These results indicate that porin accumulates in the serum of infected mice much earlier than the appearance of antibodies to porin. Thus detection of porin holds promise for early diagnosis of typhoid.     

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

The complex serologic diagnosis of typhoid fever

A parallel serological study of the blood sera of typhoid patients has been made by the methods of countercurrent immunoelectrophoresis and the indirect hemagglutination test with a view to establish the presence of soluble typhoid antigens and their corresponding antibodies. As shown in this study, the occurrence of Salmonella typhi O- and Vi-antigens is essentially higher than the content of specific antibodies in diagnostically significant titers.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.     

Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.     

A soluble 60 kilo Dalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL

Two major 60 kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl-soluble 60 kD protein is (i) structurally and antigenically distinct from the previously characterized 60 kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD mycobacterial antigen and the GroEL heat-shock protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl-GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide-rich cell wall late in the growth cycle. Chl-GroEL is identical to the Triton X-100-soluble, ocular delayed-type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl-GroEL homology to host proteins is hypothesized.     

Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.     

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.     

Regulation by light of cyclic nucleotide-dependent protein kinases and their substrates in frog rod outer segments

Cyclic nucleotides (both cAMP and cGMP) stimulate the phosphorylation of several proteins of 65-70, 50-52, 21, 13, and 12 kD in rod outer segments (ROS) of the frog retina. Subcellular fractionation showed that phosphopeptides of 67, 21, 13, and 12 kD were soluble and phosphopeptides of 69, 67, 50-52, and 12 kD were membrane associated at physiological ionic strength. Components I and II, 13 and 12 kD, respectively, are the major cyclic nucleotide-dependent phosphoproteins of ROS and have been reported to be phosphorylated in the dark and dephosphorylated in the light. Under unstimulated conditions, phosphorylated Components I and II were found in the soluble fraction. Cyclic nucleotide stimulation of phosphorylation resulted in increased phospho-Components I and II in the soluble fraction, and phospho-Component II on the membrane. Light had no effect on the phosphorylation level of soluble Components I and II, but it caused a depletion within 1 s of the membrane-bound phospho-Component II. A half-maximal decrease in membrane-bound Component II was seen at 5 x 10(5) rhodopsins bleached per outer segment. The cyclic nucleotide-dependent protein kinase(s) were found primarily in the peripheral membrane fraction of ROS proteins. 8-bromo cyclic AMP was two orders of magnitude more effective than 8-bromo cyclic GMP at stimulating Component I and II phosphorylation. An active peptide of the Walsh inhibitor of cAMP-dependent protein kinase [PKI(5-22)amide] blocked the phosphorylation with an IC50 of 10 nM. Photoaffinity labeling studies with 8-N3-cAMP and 8-N3-cGMP revealed the presence of a 52-kD band specifically labeled with 8-N3-cAMP, but no specific 8-N3-cGMP labeling. These data suggest that cyclic nucleotide-dependent protein phosphorylation in ROS occurs via the activation of a cAMP-dependent protein kinase.     

Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies

Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.     

Sensitizing properties of Brucella protein antigens synthesized in K12 Escherichia coli cells]

The sensitizing properties of brucella 31 kD and 31+15 kD protein antigens produced by Escherichia coli cell carrying and expressing the corresponding brucella genes were compared. In experiments evaluating the test of mouse feet oedema in CBA line animals the property of the 31 kD antigen preparation to induce the specific oedema effect was demonstrated on the level comparable with the one produced by the brucella outer membrane proteins preparation. The obtained data were confirmed in the experiments on adoptive transfer of prolonged type hypersensitivity via the spleen cells from the sensitized donors to intact recipient animals. The future of molecular cloning technique usage for obtaining the homogeneous stable preparations of brucella antigens with low reactivity and high specificity is discussed.     

Typhoid fever survey in two localities in Vietnam

As a part of multipurpose health survey of the population in Vietnam the antibodies against S. typhi were determined by the micromethod using haemagglutination test (O-antigen 9, 12) and agglutination test using standard H-diagnostic antigen (d). Totally 292 sera were examined, 139 from Duyen Thai village and 154 from Mai Chau. The data on vaccination against typhoid fever are recorded only in 102 persons. The positivity on Vi antibodies is very high--70% in Duyen Thai and 47% in Mai Chau. This finding is significant according to the high titres in the carriers of S. typhi. The titres of all antibodies are lower in Mai Chau area situated in mountains then in crowded lowlands of Duen Thai. The level of antibodies is decreasing with age. The frequency distribution of antibodies by age proves endemicity of the disease in area, where a large part of population is infected already before reaching 20 years of age. The effectivities of vaccination is discussed.     

Immunoelectrophoretic analysis of major component proteins in cystic fluid of Taenia solium metacestodes.

When cystic fluid of Taenia solium metacestodes (CF) was filtrated through Sephacryl S-300 Superfine, major proteins were in fractions III and IV. Major protein in fraction III was Band C protein of 150 kDa and that in fraction IV was Band N protein (Choi et al., 1990). When CF was electrophoresed in 0.9% agarose gel and reacted with anti-CF rabbit serum (RACF), two main bands, a long outer and a short inner band, were precipitated, together with 8 minor bands. RACF reacted with fraction III forming the long outer band whereas RACF formed the short inner band with fraction IV in immunoelectrophoresis (IEP). The long outer precipitin band of CF fraction III was similar to antigen B in hydatid fluid (HF) of Oriol et al. (1971), while the short inner band of CF fraction IV was similar to HF antigen 5 of Capron et al. (1967). When HF was reacted with RACF, the short inner band was immunoprecipitated without forming the long outer band. Common antigenicity between CF and HF seemed to exist in fraction IV rather than in fraction III of CF. Patient sera of neurocysticercosis reacted more frequently with fraction III than with fraction IV.     

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases

Objective. Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. Methods. PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. Results. PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. Conclusions. These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody experssion may be induced through antigen-driven mechanisms.Abbreviations mAb monoclonal antibody - PCNA proliferating cell nuclear antigen - PCNA/AK PCNA affinity purified by antibodies from patient serum AK - PCNA/TO30 PCNA purfied by mAb TO30 - PCNA/TOB7 PCNA purified by mAb TOB7 - SLE systemic lupus erythematosus     

Antigenic variation of outer membrane protein II in colonial variants of Neisseria gonorrhoeae P9

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized wtih outer membranes from colonial opacity variants in Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide, the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.     

Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin

Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.     

Discovery and identification of anti-U1-A snRNP antibody in lung cancer

There are multiple reports of autoimmune response in patients with lung cancer. To investigate whether a novel autoantibody is present in patients with lung cancer and evaluate its clinical diagnostic and prognostic value, sera from 10 patients with lung cancer and 10 normal individuals were analyzed using immunofluorescence and Western blotting. It was found that one serum sample from the patients with squamous carcinoma gave a fine speckled pattern staining in nucleus and had a high titer antinuclear autoantibody which could recognize 31 kD of nuclear protein isolated from both cancer cells and normal cells. The same patient’s serum was further used to immunoprecipitate the target antigen. The protein bands were excised from the SDS-PAGE gels and were analyzed with a Qstar Pulser I Quadrupole time-flight mass spectrometer, and the 31 kD target antigen was identified as U1-AsnRNP. To test the prevalence of anti-U1-AsnRNP antibody, sera from 93 patients including 36 squmaous carcinomas (SCC), 26 adenocarcinomas (Ad), and 31 small cell carcinomas (SCLC) were screened by Western blotting. The results demonstrated that anti-U1-A snRNP antibody was present in 50% of SCC sera, 26.9% of Ad sera and 54.8% of SCLC sera. In this paper, we report for the first time that anti-U1-AsnRNP antibody could be detected in the patients with lung cancer.