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In this report we have analysed the peripheral blood lymphocyte of several patients with chronic hepatitis B virus infection with flow cytometry. Based on the presence and absence of the HBeAb, patients were divided into two groups. In both, all the patients were HBsAg positive with normal range of serum alanine aminotranferase (23.9 ± 17.8). We have found that the immunophenotypic profiles of patients were different from healthy donors with significant decrease in CD3+ T cells, specially CD8+ T cells and a siginificant increase in the CD19+ B cells. The differences were seen in other subset of T cells (CD4+) or NK cells (CD56+/CD16+ ) and HLA-DR markers were not significant. When the phenotypic profiles of both groups were compared with each other, such changes were more dominant in group II, with HBeAb positive than in group I, with HBeAb negative. Also, we have seen a correlation between the increase of CD19+ B cells and the decrease of CD3+ T cells. No such correlation was observed with other cells.

The hepatitis B virus is a dangerous infectious agent, with an estimated 300 million carriers worldwide. Therefore, developing effective vaccine production methods is essential. One such method involves the use of transgenic plants. Several factors influence foreign gene expression in transgenic plants, including the type of genes, their chromosomal location, and the promoter used. Based on these factors, various expression cassettes can be designed for transgenic plants. In this study, we designed an expression cassette for transgenic potato plants. Promoters play a crucial role, as some are tissue-specific while others are inducible. Additionally, certain elements can enhance or silence gene expression in transgenic plants. Enhancer sequences derived from plant viruses are also utilized. To increase the expression level of HBsAg in transgenic potato plants, the tobacco etch virus (TEV) 5' untranslated region and the Patatin promoter were employed. The results demonstrated that in the highest-producing transgenic plant, the expression level of HBsAg increased threefold when both patatin and TEV were used together, compared to plants containing only the patatin promoter without TEV. Furthermore, a comparison with pCaMV combined with TEV revealed that the use of patatin and TEV resulted in a 4-5 times higher expression level. [ABSTRACT FROM AUTHOR]

Aim: In this research, we designed a direct Enzyme Linked Immunoassay method to detect Helicobacter pylori antigens in stool specimens. Background: Helicobacter pylori infection as the worldwide problem is related to many gastrointestinal disorders such as gastritis, gastric cancer, non-ulcer disease, peptic ulcer disease and duodenal ulcer. Methods: We produced and purified recombinant CagA and NapA antigens in Escherichia coli and extracted their antibodies from a panel of positive sera specimens. We designed a novel enzyme linked immunoassay direct method in combination with the whole cell for the qualitative and quantitative detection of Helicobacter pylori antigens in human stool. Assay performance was evaluated by histopathology staining and urease activity. Results: The sensitivity and specificity of assay was determined as 91.7 [95% confidence interval: 89.3-95.6%] and 93.1% [95% CI: 91.2-96.4%], respectively. Novel ELISA exhibits enhanced sensitivity and specificity of Helicobacter pylori detection in comparison with another commercially available kit. Conclusion: Combination of the recombinant antigens and whole cell of Helicobacter pylori in immunoassay designing is a new approach about early diagnosis, treatment and fallowing up of the Helicobacter pylori infected patients, especially in peptic cancer cases. [ABSTRACT FROM AUTHOR]

Background. The conventional methods for diagnosis of Vibrio cholerae are time consuming, complicated, and expensive. Development of rapid detection tests is critical for prevention and management of cholera. This study aimed to introduce two sensitive sandwich ELISAs based on avian antibodies (IgY) targeting outer membrane protein W (OmpW) and cytotoxin B (CtxB) antigens of V. cholerae. Methods. The sequences of ompW and ctxB genes were cloned into pET28a vector. Escherichia coli BL21 (DE3) was transformed with the recombinant vectors, and gene expression was induced by IPTG. The expressed proteins were purified by affinity chromatography using Ni-NTA resins. Two groups of white Leghorn chickens were immunized by recombinant proteins, and the generated antibodies were purified from egg yolks of chickens by PEG precipitation. The antibodies were used for the development of α-OmpW and α-CtxB ELISAs. Results. The expression and purification yielded 59 and 38 mg of recombinant OmpW and CtxB, respectively, per one liter of bacterial culture. PEG precipitation and purification of egg yolk antibodies yielded on average (±SD) 66.5 ± 1.80 and 50.9 ± 2.23 mg of purified α-OmpW and α-CtxB per egg, respectively. The analytical sensitivity of α-OmpW ELISA was 103 cfu/mL of V. cholerae and that of α-CtxB ELISA was 33 pg/mL of recombinant cytotoxin B. The two developed ELISAs did not show any cross-reactivity to any tested bacteria grown in common conditions. Discussion. The current study is the first report on using IgY for detection of V. cholerae. The developed ELISAs were shown to have considerable analytical sensitivity and specificity. Therefore, the assays can be one of the convenient methods for sensitive and specific detection of toxigenic V. cholerae strains in clinical and environmental samples. [ABSTRACT FROM AUTHOR]

Background: The main concern in formulation of antibodies is the intrinsic instability of these labile compounds. To evaluate the physicochemical stability of antibody in dry powder formulations, physical stability of IgG1 and a monoclonal antibody (trastuzumab) during the spray drying process was studied in a parallel study and the efficacy of some sugar based excipients in protection of antibodies was studied. Results: The SDS-PAGE analysis showed no fragmentation of antibodies after spray drying in all formulations. The secondary structure of antibodies contained 40.13 to 70.19% of β structure in dry state. Also, CD spectroscopy showed the similar secondary structure for trastuzumab after reconstitution in water. ELISA analysis and cell culture studies were conducted in order to evaluate bioactivity of monoclonal antibody. Formulations containing combination of excipients provided maximum tendency of trastuzumab to attach to the ELISA antigen (86.46% ± 2.3) and maximum bioactivity when incubated with SKBr3 cell line (the cell viability was decreased to 65.99% ± 4.6). Incubation of formulations with L929 cell line proved the biocompatibility of the excipients and non-toxic composition of formulations. Conclusion: The IgG1 and trastuzumab demonstrated similar behavior in spray drying process. The combination of excipients containing trahalose, hydroxypropyl beta cyclodextrin and beta cyclodextrin with proper ratio improved the physical and chemical stability of both IgG1 and monoclonal antibody. [ABSTRACT FROM AUTHOR]

Background: The main concern in formulation of antibodies is the intrinsic instability of these labile compounds. To evaluate the physicochemical stability of antibody in dry powder formulations, physical stability of IgG1 and a monoclonal antibody (trastuzumab) during the spray drying process was studied in a parallel study and the efficacy of some sugar based excipients in protection of antibodies was studied., Results: The SDS-PAGE analysis showed no fragmentation of antibodies after spray drying in all formulations. The secondary structure of antibodies contained 40.13 to 70.19% of β structure in dry state. Also, CD spectroscopy showed the similar secondary structure for trastuzumab after reconstitution in water. ELISA analysis and cell culture studies were conducted in order to evaluate bioactivity of monoclonal antibody. Formulations containing combination of excipients provided maximum tendency of trastuzumab to attach to the ELISA antigen (86.46% ± 2.3) and maximum bioactivity when incubated with SKBr3 cell line (the cell viability was decreased to 65.99% ± 4.6). Incubation of formulations with L929 cell line proved the biocompatibility of the excipients and non-toxic composition of formulations., Conclusion: The IgG1 and trastuzumab demonstrated similar behavior in spray drying process. The combination of excipients containing trahalose, hydroxypropyl beta cyclodextrin and beta cyclodextrin with proper ratio improved the physical and chemical stability of both IgG1 and monoclonal antibody.