Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Bacterial/*analysis ; Bacterial Proteins/analysis ; Mice ; Mice, Inbred BALB C ; Orientia tsutsugamushi/*immunology ; Species Specificity

No abstract available.
Antigens, Bacterial/*analysis ; Feces/*microbiology ; Helicobacter Infections/*diagnosis ; Helicobacter pylori/*isolation & purification ; Humans ; Immunoenzyme Techniques

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.
Antigens, Bacterial/*analysis/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Mycobacterium tuberculosis/*immunology

Although the confirmative diagnosis of typhoid fever is by culture of the causative organism, usually from blood, a serological test is still necessary to provide a more rapid method of diagnosis. The indirect fluorescent antibody test, using a Salmonella typhi Vi antigen and a FITC-conjugated rabbit anti-human polyvalent immunoglobulin, was evaluated for the diagnosis of typhoid fever. Serum specimens were collected from patients with febrile diseases on admission. Of the 32 patients with titers of 1:64 or more, 22 were confirmed to have typhoid fever by blood culture and 7 had fever of undetermined origin that was considered to be typhoid fever clinically. Three patients were diagnosed to have salmonellosis other than typhoid fever. Of the 121 patients with titers of 1:32 or less, 105 patients had non-typhoidal febrile disease, 15 patients had fever of undetermined origin, and one patient was confirmed to have typhoid fever by blood culture. When a Vi antibody titer of 1:64 or more was taken as serological evidence for the diagnosis of typhoid fever, the sensitivity and specificity were 95.7% and 97.2%, respectively. The incidence of positive test results following fever onset was 70.0% within 1 week of fever onset, 88.9% from 1 to 2 weeks, and 100% after 2 weeks. In conclusion, the Vi-indirect fluorescent antibody test(Vi-IFAT) can be employed as a useful serologic test in the diagnosis of typhoid fever.
Antigens, Bacterial/*analysis ; Fluorescent Antibody Technique/*standards ; Human ; Salmonella typhi/immunology ; Sensitivity and Specificity ; Typhoid Fever/*diagnosis

OBJECTIVETo analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank.

METHODSPCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software.

RESULTSThe complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%.

CONCLUSIONData from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

Amplified Fragment Length Polymorphism Analysis ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Bacterial Typing Techniques ; classification ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Membrane Proteins ; genetics ; Orientia tsutsugamushi ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

The changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum after treatment of syphilis were observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Until 9 to 12 months after treatment, it was seen that there was a loss of several antibodies and some diminution in their reactivity in primary, secondary and early latent syphilis, but no changes occurred in late latent and reinfected syphilis. In primary syphilis, there was a significant loss of two IgG antibodies to the treponemal antigens of molecular weights 68,500 and 47,000 at 11 months after treatment. According to our previous study, the treponemal antigen of molecular weight 68,500 was T. pallidum specific and appeared only in primary syphilis, and that of molecular weight 47,000 was one of the major antigens of T. pallidum. The reaction between serum IgG antibodies of 14 patients who had been treated for secondary, early latent and late latent syphilis 2 to 14 years ago and major antigens of T. pallidum was observed and any loss or decrease in reactivity was not discovered. From the results obtained, it was concluded that the observation of serum IgG antibody reactivity to protein antigens of T. pallidum is not helpful in evaluating the efficacy of treatment in secondary, early latent, late latent and reinfected syphilis. However, serum IgG antibodies to treponemal antigens of molecular weights 68,500 and 47,000 could possibly be useful in the assessment of the efficacy of treatment in primary syphilis.
Antibodies, Bacterial/*immunology ; Antigens, Bacterial/*analysis ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Humans ; Immunoglobulin G/*analysis ; Recurrence ; Syphilis/*diagnosis/immunology/therapy ; Time Factors ; Treponema pallidum/*immunology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

OBJECTIVEIn this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB.

METHODSSix recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models.

RESULTSTwo IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%, P<0.05). OspC B.g had the highest diagnostic value in the IgM test (AUC=0.871). Interactive effects between OspC B.a and Fla B.g could reduce the specificity of the ELISA.

CONCLUSIONThree recombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

Antigens, Bacterial ; blood ; Bacterial Proteins ; analysis ; China ; Enzyme-Linked Immunosorbent Assay ; veterinary ; Lyme Disease ; diagnosis ; Recombinant Proteins ; analysis ; Sensitivity and Specificity ; Serologic Tests ; veterinary

Lipopolysaccharide (LPS) or glycolipid antigens of Leptospira interrogans have been candidates as serogroup or serotype specific antigen. In this study, therefore, we prepared the LPS and lipid antigens from L. interrogans serovars lai, icterohaemorrhagiae, copenhageni, canicola, pomona, grippotyphosa, and a Korean isolate 30R. The LPS antigens were analyzed by a polyacrylamide gel electrophoresis and lipid antigens by thin-layer chromatography, respectively. The seroreactivity of the antigens were also examined with homologous or heterologous antisera using an enzyme-linked immunosorbent assay. The LPS antigens from serovar lai and the strain 30R were closely related but different from serovar icterohaemorrhagiae. Particularly, the LPS antigens from serovars icterohaemorrhagiae and grippotyphosa were reactive only with the homologous antisera, thus indicating serovar specificity. However, the LPS antigens of the other serovars were reactive to the heterologous antisera. The lipid antigen of serovar icterohaemorrhagiae reacted only with the homologous antisera. In contrast, lipids of other serovars reacted broadly with heterologous antisera, particularly among serovars lai, copenhageni, canicola, pomona, and the strain 30R. The results thus indicated that the LPS and lipid antigens of L. interrogans may contain serovar-specific as well as cross-reactive epitopes.
Antigens, Bacterial/*analysis ; Chromatography, Thin Layer ; Comparative Study ; Electrophoresis, Polyacrylamide Gel ; Leptospira interrogans/*chemistry/immunology ; Lipids/*analysis/immunology ; Lipopolysaccharides/*analysis/immunology ; Support, Non-U.S. Gov't