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

OBJECTIVETo develop a method of immunochromatography assay (ICA) with sensitive, specific, rapid, simple and suitable for the detection of Yersinia pestis antigen at the local laboratories.

METHODSColloidal gold labeled with the anti-F1 antibody of Yersinia pestis, was connnected with the anti-F1 antibody of Yersinia pestis to pyroxylin membrane and assembled them to the dipstick of ICA.

RESULTSResults showed that the rates of sensitivity for F1 antigen and Yersinia pestis were 1 ng/ml and 1.56 x 10(5) CFU/ml respectively. However, Yersinia pseudotuberculosis et al could not be detected by dipstick of ICA.

CONCLUSIONThe method of ICA appeared to be consistent to those of r-IHA with better specificity and sensitivity but was simple and rapid for the detection of Yersinia pestis and F1 antigen.

Bacterial Proteins ; analysis ; immunology ; Immunoassay ; methods ; Sensitivity and Specificity ; Yersinia pestis ; immunology

OBJECTIVETo identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.

METHODSTwo-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.

RESULTSA total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.

CONCLUSIONThe newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.

Bacterial Proteins ; analysis ; immunology ; Humans ; Proteomics ; Streptococcal Infections ; Streptococcus suis ; immunology ; isolation & purification ; metabolism

OBJECTIVETo recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease.

METHODSThe OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot.

RESULTSOspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity.

CONCLUSIONThe findings laid basis for the studies on early diagnosis of Lyme disease.

Antigens, Bacterial ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Blotting, Western ; Borrelia burgdorferi Group ; immunology ; Escherichia coli ; genetics ; Humans ; Lyme Disease ; diagnosis ; Polymerase Chain Reaction ; Recombinant Proteins ; analysis ; immunology

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.
Actinobacillus Infections/prevention & control ; Actinobacillus pleuropneumoniae/genetics/*immunology ; Animals ; Antibodies, Bacterial/blood ; Bacterial Proteins/analysis/*immunology ; Cytokines/analysis/blood ; Disease Models, Animal ; Female ; Hemolysin Proteins/analysis/*immunology ; Immunoglobulin A/blood/immunology ; Intestines/immunology ; Lung/cytology/immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/*immunology ; Saccharomyces cerevisiae/*genetics/immunology ; Survival Analysis ; Time Factors ; Vaccination ; Vaccines, Synthetic/administration & dosage/*immunology

OBJECTIVETo investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.

METHODSSpecific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.

RESULTSThe target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.

CONCLUSIONSCombined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.

Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; analysis ; Bacterial Proteins ; immunology ; Female ; Immunization ; Lipoproteins ; immunology ; Lung ; microbiology ; Metalloendopeptidases ; immunology ; Mice ; Mice, Inbred BALB C ; Pneumococcal Infections ; prevention & control ; Pneumococcal Vaccines ; immunology ; Saliva ; immunology

OBJECTIVEThe purpose of this study is to examine the levels of salivary SIgA and serum IgG induced by pcDNA3-pac and pcDNA3-gtfB immunization, so as to testify the antigenity of the two gene vaccines.

METHODS36 28-day-old Wistar rats were divided into 6 groups, among which 3 experimental groups were vaccinated with pcDNA3-pac, pcDNA3-gtfB or pcDNA3-pac combined with pcDNA3-gtfB, respectively, one positive control was vaccinated with inactive whole cell of S. mutans JBP and other two negative controls were injected with the vector pcDNA3 or PBS buffer, respectively. All vaccines and materials were delivered with 100 micrograms by submandibular gland injection for 3 times. Then the restricted bacterial model of rat was constructed. Following that all rats were fed with cariogenic diet Keyes 2000 for 3 months, saliva and serum samples were collected to assay SIgA or IgG levels by ELASA.

RESULTSThe salivary S-IgA levels both in pcDNA3-pac combined with pcDNA3-gtfB group and inactive S. mutans cell group were higher than others (P < 0.01). In groups of pcDNA3 and PBS buffer, they were lowest (P < 0.01). The serum IgG levels in the three experimental groups and positive control were higher than that in negative control (P < 0.05). It was important that salivary SIgA in groups of gene vaccine and inactive S. mutans vaccination reached its peak at the 11th week after the first inoculation and kept until the end of the study.

CONCLUSIONBoth pcDNA3-pac and pcDNA3-gtfB can express immunogenic protein and induce immune responses of mucosal and humoral immune system in gnobobiotic rats. It is also indicated that the joint gene vaccines immunization is an optimal choice for anticaries strategy.

Animals ; Antibodies, Bacterial ; analysis ; blood ; Dental Caries ; prevention & control ; Female ; Glucosyltransferases ; immunology ; Immunoglobulin A, Secretory ; immunology ; Immunoglobulin G ; analysis ; Male ; Membrane Proteins ; immunology ; Random Allocation ; Rats ; Rats, Wistar ; Saliva ; immunology ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; immunology ; Vaccination ; Vaccines, DNA ; immunology

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

OBJECTIVETo reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body.

METHODSAccording to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s.

RESULTThe expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope.

CONCLUSIONLipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

Amino Acid Sequence ; Animals ; Antigens, Bacterial ; genetics ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Bacterial Vaccines ; immunology ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Immune Sera ; immunology ; Leptospira interrogans ; genetics ; immunology ; ultrastructure ; Lipoproteins ; genetics ; immunology ; metabolism ; Microscopy, Immunoelectron ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sequence Analysis, DNA ; Vaccines, DNA ; immunology

OBJECTIVETo investigate the immunoregulatory effects of pertussis protein on airway inflammatory, IFN-gamma/IL-4 ratio in bronchoalveolar lavage fluids(BALF) and airway hyperresponsiveness (AHR) in the sensitized mice.

METHODSThe sensitized mice were reexposed to ovalbumin and the airway response to methacholine injection was monitored. Inflammatory cells and cytokines IFN-gamma/IL-4 ratio in BALF were measured. Lung tissue specimens were collected for histological examination.

RESULTIntramuscular injection or intranasal instillation of pertussis protein inhibited changes in lung resistance and lung dynamic compliance, upregulated IFN-gamma/IL-4 ratio and decreased eosinophil accumulation in a dose-dependent manner. Pathological examination showed that goblet cell hyperplasia and inflammatory cells infiltration in lung tissue were suppressed by pertussis protein.

CONCLUSIONPertussis protein inhibits the inflammation and regulates the function of lungs in asthma mice, suggesting its potential application in treatment of asthma.

Albumins ; Animals ; Asthma ; chemically induced ; immunology ; therapy ; Bacterial Proteins ; immunology ; pharmacology ; Bacterial Toxins ; immunology ; pharmacology ; Bronchoalveolar Lavage Fluid ; chemistry ; Interferon-gamma ; analysis ; Interleukin-4 ; analysis ; Male ; Methacholine Chloride ; Mice ; Mice, Inbred ICR