Corrigendum to "Development of IgY-Based Sandwich ELISA as a Robust Tool for Rapid Detection and Discrimination of Toxigenic Vibrio cholera".

[This corrects the article DOI: 10.1155/2018/4032531.].

Development of IgY-Based Sandwich ELISA as a Robust Tool for Rapid Detection and Discrimination of Toxigenic Vibrio cholerae.

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.

A comparative study on the physicochemical and biological stability of IgG1 and monoclonal antibodies during spray drying process.

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 ß 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.

Nano-adjuvant based on silk fibroin for the delivery of recombinant hepatitis B surface antigen.

Nanotechnology has a vital role in vaccine development. Nano-adjuvants, as robust delivery systems, could stimulate immune responses. Using nanoparticles (NPs) in vaccine formulations enhances the target delivery, immunogenicity, and stability of the antigens. Herein, silk fibroin nanoparticles (SFNPs) were used as a nano-adjuvant for delivering recombinant hepatitis B surface antigen (HBsAg). HBsAg was loaded physically and chemically on the surface of SFNPs. The HBsAg-loaded SFNPs had a spherical morphology. The in vitro release studies showed that HBsAg had a continuous and slow release from SFNPs during 56 days. During this time, ∼45.6% and 34.1% HBsAg was released from physical-SFNPs and chemical-SFNPs, respectively. HBsAg-loaded SFNPs were also stable for six months with slight changes in the size, surface charge, and morphology. The results of circular dichroism (CD) and fluorescence spectroscopy indicated that the released HBsAg preserved the native secondary and tertiary structures. The quantitative cellular uptake study also showed that physical-SFNPs were taken up more into J774A.1 macrophage cells than chemical-SFNPs. After 28 and 56 days post-injection, the immunogenicity studies showed that the specific total IgG, IgG1, and IgG2a levels against HBsAg were significantly higher in the physically loaded group than in the chemically loaded group and commercial hepatitis B vaccine. IgG2a levels were detected only in mice immunized with physical-SFNPs. However, the low levels of IL-4 and IFN-γ were produced in all vaccinated groups and differences in mean values were not significant compared with control groups. Results indicated an improvement in the levels of anti-HBsAg IgG in mice immunized with the physical-SFNPs group compared to other groups.

Designing a novel ELISA method based on CagA, NapA recombinant antigens to increase sensitivity and specificity of Helicobacter pylori whole cell antigen detection.

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.

High avidity anti-integrase antibodies discriminate recent and non-recent HIV infection: Implications for HIV incidence assay.

Estimation of HIV incidence provides real-time information of HIV transmission trends for decision makers. Anti-integrase antibodies are the last ones produced during seroconversion and presence of high-avidity anti-integrase antibodies indicates the chronicity of HIV infection. This study aimed to evaluate the performance of these antibodies in discriminating of recent from non-recent HIV infection. For this purpose, different ELISA formats were developed to detect high-avidity anti-integrase antibodies in a commercially available performance panel, and the best assay was selected for further evaluation. The false recent rate of the selected assay was evaluated in a panel of Iranian patients and compared to two commercial assays, BED-EIA and LAg-Avidity. While the false recent rate of the developed assay was 3.8%, it was 14.1% and 1.3% for BED-EIA and LAg-Avidity, respectively. To our knowledge, this is the first report to study the performance of high-avidity anti-integrase antibodies for classification of HIV infection. The preliminary results showed that the specificity of the newly developed assay is markedly higher than BED-EIA and is comparable with LAg-Avidity. The promising results point to the potential use of anti-integrase antibodies as a biomarker in HIV incidence laboratory tests or algorithms. The developed assay needs further evaluation in future.

Development of an integrase-based ELISA for specific diagnosis of individuals infected with HIV.

Currently, enzyme immunoassays (EIAs) are the most common immunological diagnostic methods that are used as the screening tool in HIV detection. Among all three major genes of HIV, the products of gag and env are usually used in EIAs (ELISAs and rapid tests). Hence, the presence of cross reacting antibodies against these antigens leads to the appearance of repetitive false positive results in screening tests. Re-testing the primary reactive samples with EIAs using other HIV antigens can considerably reduce the rate of false positive results. The products of pol gene may act as an appropriate candidate in this context. Integrase is a conserved and immunogenic product of HIV, encoded by the pol gene. The aim of this research was to determine the sensitivity and specificity of an ELISA detecting integrase antibodies. Recombinant integrase was produced in Escherichia coli to develop the integrase-based ELISA. Assay performance was evaluated by HIV positive and negative sera and an HIV panel of BBI (PRB-601). The sensitivity and specificity of assay was determined as 96.7 [95% confidence interval: 91.3-98.9%] and 100% [95% CI: 96.1-100%], respectively. High specificity of this assay may suggest its possible use in the detection of HIV.

Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications.

BACKGROUND: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. OBJECTIVES: The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco. MATERIALS AND METHODS: A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N- to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. RESULTS: The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications. CONCLUSIONS: By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible.

Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor.

Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

HCV core protein immunization with Montanide/CpG elicits strong Th1/Th2 and long-lived CTL responses.

An efficient vaccine against Hepatitis C virus (HCV) infection requires induction of strong humoral and cellular responses against viral proteins. We evaluated the immunogenicity of HCV core protein (HCVcp), a prime vaccine candidate, formulated in various human compatible adjuvants. An Escherichia coli-expressed HCVcp, purified in native conditions was used for murine immunization in separate groups of: free HCVcp (Ag), Ag+C/IFA (Freunds), Ag+CpG, Ag+M720 (Montanide ISA 720), Ag+F127 (Pluronic acid) and cocktails of Ag+F127+CpG and Ag+M720+CpG. Mice immunized with M720(+CpG) developed the highest HCVcp-specific titers of total IgG, IgG1, 2a, 2b, and that of IFN-gamma and IL-4 cytokines compared to all other groups. HCVcp-specific-CTLs against relevant MHC class I peptides were detected only for Ag+M720+CpG, Ag+M720, and Ag+CpG groups and could be blocked by antimouse-CD8 antibodies. While CTLs were stable, only F127 formulated groups demonstrated detectable IgG antibodies one year post-immunization. Hence, HCVcp formulated in M720 (with a synergistic effect by inclusion of CpG) could induce balanced and strong Th1/Th2 responses with long-lived CD4(-)CD8(+) CTLs.

Compositional changes of PBL population in patients with chronic hepatitis B virus infection

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 CD(3)(+) T cells, specially CD(8)(+) T cells and a significant increase in the CD(19)(+) B cells. The differences were seen in other subset of T cells (CD(4)(+)) or NK cells (CD(56)(+)/CD(16)(+)) 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 CD(19)(+) B cells and the decrease of CD CD(3)(+) T cells. No such correlation was observed with other cells.