Evaluation of an in silico predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of Chlamydia trachomatis infections

<p>Abstract</p> <p>Background</p> <p>The OmcB protein is one of the most immunogenic proteins in <it>C. trachomatis </it>and <it>C. pneumoniae </it>infections. This protein is highly conserved leading to serum cross reactivity between the various chlamydial species. Since previous studies based on recombinant proteins failed to identify a species specific immune response against the OmcB protein, this study evaluated an <it>in silico </it>predicted specific and immunogenic antigen from the OmcB protein for the serodiagnosis of <it>C. trachomatis </it>infections.</p> <p>Results</p> <p>Using the ClustalW and Antigenic programs, we have selected two predicted specific and immunogenic regions in the OmcB protein: the N-terminal (Nt) region containing three epitopes and the C-terminal (Ct) region containing two epitopes with high scores. These regions were cloned into the PinPoint Xa-1 and pGEX-6P-1 expression vectors, incorporating a biotin purification tag and a glutathione-S-transferase tag, respectively. These regions were then expressed in <it>E. coli</it>. Only the pGEX-6P-1 has been found suitable for serological studies as its tag showed less cross reactivity with human sera and was retained for the evaluation of the selected antigens. Only the Ct region of the protein has been found to be well expressed in <it>E. coli </it>and was evaluated for its ability to be recognized by human sera. 384 sera were tested for the presence of IgG antibodies to <it>C. trachomatis </it>by our in house microimmunofluorescence (MIF) and the developed ELISA test. Using the MIF as the reference method, the developed OmcB Ct ELISA has a high specificity (94.3%) but a low sensitivity (23.9). Our results indicate that the use of the sequence alignment tool might be useful for identifying specific regions in an immunodominant antigen. However, the two epitopes, located in the selected Ct region, of the 24 predicted in the full length OmcB protein account for approximately 25% of the serological response detected by MIF, which limits the use of the developed ELISA test when screening <it>C. trachomatis </it>infections.</p> <p>Conclusion</p> <p>The developed ELISA test might be used as a confirmatory test to assess the specificity of serological results found by MIF.</p

Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of Chlamydophila pneumoniae IgA antibodies

<p>Abstract</p> <p>Background</p> <p>Serologic diagnosis of <it>Chlamydophila pneumoniae </it>(Cpn) infection routinely involves assays for the presence of IgG and IgM antibodies to Cpn. Although IgA antibodies to Cpn have been found to be of interest in the diagnosis of chronic infections, their significance in serological diagnosis remains unclear. The microimmunofluorescence (MIF) test is the current method for the measurement of Cpn antibodies. While commercial enzyme linked immunosorbent assays (ELISA) have been developed, they have not been fully validated. We therefore evaluated and optimized a commercial ELISA kit, the SeroCP IgA test, for the detection of Cpn IgA antibodies.</p> <p>Methods</p> <p>Serum samples from 94 patients with anti-Cpn IgG titers ≥ 256 (study group) and from 100 healthy blood donors (control group) were tested for the presence of IgA antibodies to Cpn, using our in-house MIF test and the SeroCP IgA test. Two graph receiver operating characteristic (TG-ROC) curves were created to optimize the cut off given by the manufacturer.</p> <p>Results</p> <p>The MIF and SeroCP IgA tests detected Cpn IgA antibodies in 72% and 89%, respectively, of sera from the study group, and in 9% and 35%, respectively, of sera from the control group. Using the MIF test as the reference method and the cut-off value of the ELISA test specified by the manufacturer for seropositivity and negativity, the two tests correlated in 76% of the samples, with an agreement of Ƙ = 0.54. When we applied the optimized cut-off value using TG-ROC analysis, 1.65, we observed better concordance (86%) and agreement (0.72) between the MIF and SeroCP IgA tests.</p> <p>Conclusion</p> <p>Use of TG-ROC analysis may help standardize and optimize ELISAs, which are simpler, more objective and less time consuming than the MIF test. Standardization and optimization of commercial ELISA kits may result in better performance.</p

Antibiosis and bmyB Gene Presence As Prevalent Traits for the Selection of Efficient Bacillus Biocontrol Agents against Crown Gall Disease

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains

Synthèse d’arômes et désacidification d’une huile acide en milieu sans solvant

La neutralisation partielle de l’huile de grignon a été réalisée par estérification des acides gras libres, catalysé par le lipozyme™ en milieu sans solvant organique et à 40 °C. Le taux d’acidité de cette huile est passé alors de 30 à 13%. Le même enzyme a été utilisé pour synthétiser des arômes naturels en absence de solvant organique

Synthèse d’arômes et désacidification d’une huile acide en milieu sans solvant

La neutralisation partielle de l’huile de grignon a été réalisée par estérification des acides gras libres, catalysé par le lipozyme™ en milieu sans solvant organique et à 40 °C. Le taux d’acidité de cette huile est passé alors de 30 à 13%. Le même enzyme a été utilisé pour synthétiser des arômes naturels en absence de solvant organique

Two Novel Bacillus Strains (subtilis and simplex Species) with Promising Potential for the Biocontrol of Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch of Wheat

Two novel Algerian field-collected isolates were selected for their antifungal activity against Zymoseptoria tritici (teleomorph Mycosphaerella graminicola). The novel strains, termed Alg.24B1 and Alg.24B2, were identified as Bacillus subtilis and Bacillus simplex since their respective nucleotide sequences of the 16S rRNA gene were 100% and 99.93% identical to those of B. subtilis and B. simplex, respectively. The antifungal activities of Alg.24B1 and Alg.24B2 were evaluated by the well diffusion method and compared to those of other Bacillus species. The maximum activity was obtained after two days of confrontation of the bacterial strain supernatants with the fungus for Alg.24B1 and three days for Alg.24B2. Furthermore, the metabolites responsible for the antifungal activity of both strains were detected by the investigation of either gene presence (PCR) or molecule production (activity detection of lytic enzymes and HPLC detection of lipopeptides). Overall, this study showed that in addition to their ability to produce lytic enzymes (protease and β-glucanase), both strains coproduce three types of lipopeptides viz. surfactin, iturin, and fengycin. Thus, the biofungicide activity of both strains may be a result of a combination of different mechanisms. Therefore, they had a great potential to be used as biocontrol agents to effectively manage septoria tritici blotch of wheat (STB)

Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies-1

Bution of SeroCP IgA index in relation to MIF IgA antibody seropositivity in the control group. The continuous lines are the manufacturer's cut off indices, and the discontinuous lines are the optimized cut off values.<p><b>Copyright information:</b></p><p>Taken from "Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies"</p><p>http://www.biomedcentral.com/1471-2334/8/98</p><p>BMC Infectious Diseases 2008;8():98-98.</p><p>Published online 26 Jul 2008</p><p>PMCID:PMC2515311.</p><p></p

Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies-2

<p><b>Copyright information:</b></p><p>Taken from "Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies"</p><p>http://www.biomedcentral.com/1471-2334/8/98</p><p>BMC Infectious Diseases 2008;8():98-98.</p><p>Published online 26 Jul 2008</p><p>PMCID:PMC2515311.</p><p></p

Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies-0

E differences between OD 450 nm values were less than 20%.<p><b>Copyright information:</b></p><p>Taken from "Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of IgA antibodies"</p><p>http://www.biomedcentral.com/1471-2334/8/98</p><p>BMC Infectious Diseases 2008;8():98-98.</p><p>Published online 26 Jul 2008</p><p>PMCID:PMC2515311.</p><p></p